Prefusion-stabilized hmpv f proteins

Abstract

To provide engineered human metapneumovirus (hMPV) having enhanced conformational stability and / or antigenicity.SOLUTION: Provided is a nucleic acid molecule comprising a nucleotide sequence encoding an amino acid sequence of an engineered protein comprising metapneumovirus (MPV) F protein ectodomain having at least 90% identity to a plurality of specific amino acid sequences, where the engineered protein comprises engineered disulfide bonds comprising a paired cysteine substitutions corresponding to T127C and N153C, and / or L110C and N322C to any one of a plurality of specific amino acid sequences, and the nucleic acid molecule is an mRNA molecule. A pharmaceutical composition comprising the nucleic acid molecule is also provided.SELECTED DRAWING: None

Description

Technical Field

[0001] Cross - Reference to Related Applications This application claims the benefit of priority of U.S. Provisional Application No. 63 / 089,978, filed on October 9, 2020, the entire content of which is incorporated herein by reference.

[0002] Reference to a Sequence Listing This application includes a sequence listing submitted in ASCII format via EFS - Web, the entire content of which is incorporated herein by reference. A copy of the ASCII created on October 7, 2021, is named UTFBP1250WO_ST25.txt and is 50.6 bytes in size.

[0003] 1. Technical Field The present disclosure generally relates to the fields of medicine, virology, immunology, and protein engineering. More specifically, the present disclosure relates to engineered human metapneumovirus (hMPV) F protein and its use in drug design and vaccine formulation.

Background Art

[0004] 2. Description of Related Art Human metapneumovirus (hMPV) is a respiratory virus of the family Pneumoviridae (pneumovirus) that has been circulating in humans for at least half a century prior to its discovery in 2001 (van den Hoogen et al., 2001). Infections occur almost ubiquitously by the age of 5, and the burden of reinfection persists throughout life (van den Hoogen et al., 2001). However, infants (6 - 12 months), the elderly, and immunocompromised individuals are at increased risk of hospitalization due to more severe diseases such as pneumonia and bronchitis (Deffrasnes et al., 2007). Despite the disease burden presented by hMPV, there are no vaccines or therapeutics approved for prevention or treatment. As a member of the family Pneumoviridae (recently upclassified from a subfamily within the family Paramyxoviridae), hMPV is an enveloped negative - strand RNA virus. Viruses within this family encode three membrane proteins expressed on the surface. In the case of hMPV, these are the small hydrophobic (SH), attachment (G), and fusion (F) proteins (Shafagati & Williams, 2018).

[0005] As a class I viral fusion glycoprotein, hMPV F is initially translated as a single polypeptide precursor (F0). Proteolytic cleavage converts F0 into disulfide-linked F2 and F1 subunits (Figure 1A). Then, three F2 / F1 heterodimers associate to form a metastable prefusion trimer that constitutes the active protein. In cell culture, this proteolytic activation can be accomplished by the addition of trypsin, which cleaves the protein at a monobasic cleavage site (van den Hoogen et al., 2001, Skiadopoulos et al., 2006, Schickli et al., 2005). During natural infection, hMPV F0 is cleaved by trypsin-like extracellular serine proteases such as TMPRSS2, but the extent to which this occurs within the producing cells as opposed to the target cells has not been clearly defined (Shirogane et al., 2008). The N-terminus of the mature F1 subunit contains a hydrophobic sequence called the fusion peptide, which is located within the internal cavity of the prefusion F trimer (Battles et al., 2017). For other class I fusion proteins such as human respiratory syncytial virus F (RSV F) and influenza hemagglutinin (HA), the trimers have been shown to be unstable and capable of transiently expanding and opening, or "breathing" (Bangaru et al., 2019, Watanabe et al., 2019, Gilman et al., 2019). Recently, human antibodies targeting the trimer interface of hMPV F have been described, suggesting that prefusion hMPV F undergoes this transient opening in vivo (Huang et al., 2020). To facilitate membrane fusion, the metastable prefusion F protein undergoes substantial conformational changes to release the fusion peptide and extend it into the host cell membrane. This unstable prehairpin intermediate refolds back onto itself to form a highly stable six-helix bundle composed of trimers of the N-terminal and C-terminal heptad repeats (HRA and HRB, respectively) in the so-called postfusion conformation (Mas et al., 2016).Considering its essential role in virus entry, vaccine candidates against hMPV generally include the F protein. In other words, potential vaccination strategies against hMPV target its fusion (F) glycoprotein, which is essential for viral infection. However, there remains a need for stabilized F proteins that can be used to identify drug candidates and to stimulate an effective immune response against the F protein.

SUMMARY OF THE INVENTION

[0006] Accordingly, provided herein are pre-fusion conformational variants of thermostable hMPV F. By introducing disulfide bonds through the introduction of pairs of cystine mutations, the protein expression level and thermostability were improved. Individual hydrophobicity, electrostatic interaction, and charge reduction mutations were also beneficial. In addition, by combining multiple beneficial mutations, the desired protein properties were further improved.

[0007] In one embodiment, as used herein, there is provided an engineered protein comprising an external domain of the metapneumovirus (MPV) F protein that has at least 90% identity to (i) amino acids 19 - 489 of any one of SEQ ID NOs: 1, 2, and 4 - 7, or (ii) amino acids 19 - 484 of SEQ ID NO: 3, wherein the engineered protein comprises at least one mutation relative to any one of the sequences of SEQ ID NOs: 1 - 7, and the at least one mutation comprises a substitution at a position corresponding to K166, N342, A / D185, K188, T49, V262, H435, E26, G439, N46, L158, A161, L50, V162, E51, R163, V104, N457, L110, N322, A113, D336, A116, A338, A140, A147, S291, S443, S293, S444, S355, V442, T365, V463, S22, G53, V169, E305, L302, V47, A159, T127, N153, G121, I / F258, G106, A107, T160, I128, A190, V118, Q426, L165, V191, S149, I137, V / I122, S192, T317, L105, L134, A117, S347, G261, I268, S470, L473, S265, L460, F48, Q455, V231, A374, I217, S376, G366, S194, L219, A344, A86, T114, V148, D461, L66, L73, N145, Q195, E453, and / or H368. In some aspects, the engineered protein has at least 95% identity to (i) amino acids 19 - 489 of any one of SEQ ID NOs: 1, 2, and 4 - 7, or (ii) amino acids 19 - 484 of SEQ ID NO: 3. SEQ ID NO: 1 corresponds to the BV - 115 variant sequence. SEQ ID NO: 2 corresponds to the JSM - 1147 variant sequence. SEQ ID NO: 3 corresponds to the DW - 1 variant sequence. SEQ ID NO: 4 corresponds to the F protein of the hMPV A1 NL / 1 / 00 strain (GenBank: AAK62968.2). SEQ ID NO: 5 corresponds to the F protein of the hMPV A2 NL / 00 / 17 strain (GenBank: ACJ70115.1).SEQ ID NO: 6 corresponds to the F protein of the hMPV B1 NL / 1 / 99 strain (GenBank: AY525843.1). SEQ ID NO: 7 corresponds to the F protein of the hMPV B2 TN / 99 / 419 strain (GenBank: AAS92882.1).

[0008] In some embodiments, the engineered protein comprises a proline substitution corresponding to A / D185P. In some embodiments, the engineered protein comprises a substitution corresponding to RQSR (residues 99 - 102 of any one of SEQ ID NOs: 4 - 7; SEQ ID NO: 9) to RRRR (residues 99 - 102 of any one of SEQ ID NOs: 1 - 3; SEQ ID NO: 10). In some embodiments, the engineered protein comprises a proline substitution corresponding to A / D185P and a substitution corresponding to RQSR (residues 99 - 102 of any one of SEQ ID NOs: 4 - 7; SEQ ID NO: 9) to RRRR (residues 99 - 102 of any one of SEQ ID NOs: 1 - 3; SEQ ID NO: 10). In some embodiments, the engineered protein comprises a substitution corresponding to GGGGSGGGGSR (SEQ ID NO: 8) from residues 87 - 104 of any one of SEQ ID NOs: 1 - 7.

[0009] In some embodiments, the engineered protein comprises engineered disulfide bonds comprising pairs of cysteine substitutions corresponding to E26C and G439C, N46C and L158C, T49C and A161C, L50C and V162C, E51C and R163C, E51C and K166C, V104C and N457C, L110C and N322C, A113C and D336C, A116C and A338C, A140C and A147C, S291C and S443C, S293C and S443C, S293C and S444C, S355C and V442C, T365C and V463C, S22C and H435C, G53C and K166C, G53C and V169C, E305C and N457C, S291C and L302C, V47C and A159C, T127C and N153C, G121C and I / F258C, F48C and T160C, and / or T365C and Q455C. In some embodiments, the engineered protein comprises engineered disulfide bonds comprising pairs of cysteine substitutions corresponding to A116C and A338C, T365C and V463C, T127C and N153C, T365C and Q455C, V104C and N457C, L110C and N322C, or A140C and A147C. In some embodiments, the engineered protein comprises engineered disulfide bonds comprising pairs of cysteine substitutions corresponding to A140C and A147C. In some embodiments, the engineered protein comprises engineered disulfide bonds comprising pairs of cysteine substitutions corresponding to V104C and N457C. In some embodiments, the engineered protein comprises engineered disulfide bonds comprising pairs of cysteine substitutions corresponding to L110C and N322C. In some embodiments, the engineered protein comprises engineered disulfide bonds comprising pairs of cysteine substitutions corresponding to T365C and V463C.

[0010] In some embodiments, the engineered protein comprises substitutions at positions corresponding to L219 and / or V231. In some embodiments, the engineered protein comprises substitutions corresponding to L219K and / or V231I. In some embodiments, the engineered protein comprises a pair of cysteine substitutions corresponding to T127C and N153C. In some embodiments, the engineered protein comprises a pair of cysteine substitutions corresponding to L110C and N322C. In some embodiments, the engineered protein comprises a pair of cysteine substitutions corresponding to A140C and A147C. In some embodiments, the engineered protein comprises a substitution corresponding to G366S.

[0011] In some embodiments, the engineered protein comprises substitutions at positions corresponding to Q426, T49, L187, L473, and / or S347. In some embodiments, the engineered protein comprises substitutions corresponding to Q426W, T49E, L187F, L473F, and / or S347Q.

[0012] In some embodiments, the engineered protein comprises engineered disulfide bonds that include a pair of cysteine substitutions corresponding to A116C and A338C. In some embodiments, the engineered protein comprises engineered disulfide bonds that include a pair of cysteine substitutions corresponding to T365C and V463C. In some embodiments, the engineered protein comprises engineered disulfide bonds that include a pair of cysteine substitutions corresponding to T127C and N153C. In some embodiments, the engineered protein comprises engineered disulfide bonds that include a pair of cysteine substitutions corresponding to T365C and Q455C. In some embodiments, the engineered protein comprises at least one additional engineered disulfide bond.

[0013] In some embodiments, the engineered protein comprises a cavity-filling substitution at a position corresponding to G106, A107, T160, L158, I128, A190, V118, Q426, L165, V191, T160, S149, I137, S149, V169, N46, T49, V / I122, S192, T317, V162, L105, L134, A117, S347, V47, G261, I268, S470, V231, A374, I217, and / or S355. In some embodiments, the engineered protein comprises a cavity-filling substitution at a position corresponding to L105, V118, I137, S149, L158, L165, or Q426. In some embodiments, the engineered protein comprises a substitution corresponding to L105I or L105W. In some embodiments, the engineered protein comprises a substitution corresponding to L158W. In some embodiments, the engineered protein comprises a substitution corresponding to V118F or V118M. In some embodiments, the engineered protein comprises a substitution corresponding to Q426W. In some embodiments, the engineered protein comprises a substitution corresponding to L165F. In some embodiments, the engineered protein comprises a substitution corresponding to S149V or S149I. In some embodiments, the engineered protein comprises a substitution at a position corresponding to I137. In some embodiments, the engineered protein comprises a substitution corresponding to I137L.

[0014] In some embodiments, the engineered protein comprises a cavity-filling substitution selected from the group consisting of G106W, A107F, T160M, L158W, I128F, A190M, V118F, V118M, Q426W, L165F, V191I, T160V, S149V, I137L, S149I, V169I, N46V, T49I, V / I122L, S192L, T317L, V162F, V162W, L105I, L105F, L105W, L134I, A117M, S347M, S347K, S347Q, V47M, G261M, I268M, S470Y, V231I, A374V, I217V, and / or S355F.

[0015] In some embodiments, the engineered protein comprises a proline substitution selected from the group consisting of A86P, A107P, A113P, T114P, V148P, S443P, D461P, L130P, L141P, K142P, E146P, L151P, N153P, V162P, A / D185P, D186P, L187P, K188P, N342P, and A344P.

[0016] In some embodiments, the engineered protein comprises a substitution at a position corresponding to S376, G366, and / or S194. In some embodiments, the engineered protein comprises a substitution corresponding to S376T, G366S, and / or S194Q.

[0017] In some embodiments, the engineered protein comprises a substitution at a position corresponding to K166. In some embodiments, the engineered protein comprises a substitution corresponding to K166E.

[0018] In some embodiments, the engineered protein comprises a substitution at a position corresponding to H435 that modulates pH sensitivity. In some embodiments, the engineered protein comprises a substitution corresponding to H435E, H435D, or H435N.

[0019] In some embodiments, the engineered protein comprises an electrostatic interaction substitution at a position corresponding to L66, L73, N145, Q195, E453, L66, K188, H368, D461, T49, and / or V262. In some embodiments, the engineered protein comprises a substitution corresponding to L66N, L73E, N145E, Q195K, E453Q, L66D, K188R, H368R, D461E, T49E, and / or V262D.

[0020] In some embodiments, the engineered protein comprises a substitution corresponding to L110C, T127C, A140C, A147C, N153C, L219K, V231I, N322C, T365C, E453Q, and / or V463C.

[0021] In some embodiments, the engineered protein comprises substitutions corresponding to T127C, N153C, A185P, T365C, V463C, L219K, and V231I. In some embodiments, the engineered protein comprises substitutions corresponding to T127C, N153C, A185P, T365C, V463C, L219K, V231I, and a substitution from RQSR (residues 99 - 102 of any one of SEQ ID NOs: 1 - 7) to RRRR (SEQ ID NO: 10). In some embodiments, the engineered protein comprises substitutions corresponding to T127C, N153C, T365C, V463C, L219K, and V231I. In some embodiments, the engineered protein comprises a polypeptide sequence that is at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 14 or 16.

[0022] In some embodiments, the engineered protein comprises substitutions corresponding to L110C, T127C, A140C, A147C, N153C, A185P, L219K, V231I, N322C, T365C, N368H, E453Q, and V463C. In some embodiments, the engineered protein comprises substitutions corresponding to L110C, T127C, A140C, A147C, N153C, A185P, L219K, V231I, N322C, T365C, N368H, E453Q, V463C, and a substitution from RQSR (residues 99 - 102 of any one of SEQ ID NOs: 1 - 7) to RRRR (SEQ ID NO: 10). In some embodiments, the engineered protein comprises substitutions corresponding to L110C, T127C, A140C, A147C, N153C, L219K, V231I, N322C, T365C, N368H, E453Q, and V463C. In some embodiments, the engineered protein comprises a polypeptide sequence that is at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 15 or 17.

[0023] In some embodiments, the engineered protein comprises a substitution from residues 87 - 104 of any one of SEQ ID NOs: 1 - 7 to GGGGSGGGGSR (SEQ ID NO: 8).

[0024] In some embodiments, the engineered protein may contain any combination of the substitutions disclosed in Table 1. In some embodiments, the engineered protein may contain any combination of the substitutions disclosed in Table 1 in combination with the proline substitution corresponding to A / D185P. In some embodiments, the engineered protein may contain any combination of the substitutions disclosed in Table 1 in combination with the substitution corresponding to RQSR (residues 99-102 of any one of SEQ ID NOs: 4-7; SEQ ID NO: 9) to RRRR (residues 99-102 of any one of SEQ ID NOs: 1-3; SEQ ID NO: 10). In some embodiments, the engineered protein may contain any combination of the substitutions disclosed in Table 1 in combination with the proline substitution corresponding to A / D185P and the substitution corresponding to RQSR (residues 99-102 of any one of SEQ ID NOs: 4-7; SEQ ID NO: 9) to RRRR (residues 99-102 of any one of SEQ ID NOs: 1-3; SEQ ID NO: 10).

[0025] In some embodiments, the engineered protein contains a combination of at least one engineered disulfide bond, at least one cavity-filling substitution, and at least one proline substitution.

[0026] In some embodiments, the engineered protein has at least 95% identity to the amino acid sequence of any one of SEQ ID NOs: 1-3. In some embodiments, the engineered hMPV F protein ectodomain has 95% identity to SEQ ID NO: 3.

[0027] In some embodiments, the engineered protein is fused or conjugated to a trimerization domain. In some embodiments, the engineered protein is fused or conjugated to a trimerization domain. In some embodiments, the trimerization domain contains the trimerization domain of T4 fibritin.

[0028] In some embodiments, the engineered protein is fused or conjugated to a transmembrane domain. In some embodiments, the engineered protein is fused to a transmembrane domain. In some embodiments, the transmembrane domain comprises the transmembrane domain of the metapneumovirus (MPV) F protein.

[0029] In some embodiments, the engineered protein comprises an N-terminal signal sequence. In some embodiments, the N-terminal signal sequence is MSWKVMIIISLLITPQHG (residues 1-18 of SEQ ID NO: 6 or 7; SEQ ID NO: 11). In some embodiments, the N-terminal signal sequence is MSWKVVIIFSLLITPQHG (residues 1-18 of any one of SEQ ID NOs: 1-5).

[0030] In one embodiment, provided herein is a trimer of an engineered metapneumovirus (MPV) F protein comprising at least one subunit as described in any one of the embodiments of the engineered protein herein. In some embodiments, the trimer is stabilized in the pre-fusion conformation as compared to the trimer of the wild-type metapneumovirus (MPV) F subunit. In some embodiments, the trimer comprises at least one engineered disulfide bond between subunits. In some embodiments, at least one engineered disulfide bond between subunits is formed by substitutions corresponding to S316C and D421C.

[0031] In one embodiment, herein a pharmaceutically acceptable carrier; and (i) an engineered protein as described in any one of the embodiments of the engineered protein herein, or (ii) an engineered trimer as described in any one of the embodiments of the engineered trimer is provided. In some embodiments, the pharmaceutical composition further comprises an adjuvant.

[0032] In one embodiment, provided herein is a nucleic acid molecule comprising a nucleotide sequence encoding the amino acid sequence of the engineered protein described in any one of the embodiments of the engineered protein herein. In some embodiments, the nucleic acid comprises a DNA expression vector. In some embodiments, the nucleic acid comprises mRNA.

[0033] In one embodiment, provided herein is a method for preventing metapneumovirus (MPV) infection or a disease associated with metapneumovirus infection in a subject, the method comprising administering to the subject an effective amount of the pharmaceutical composition described in any one of the embodiments of the pharmaceutical composition herein or the nucleic acid molecule described in any one of the embodiments of the nucleic acid molecule herein.

[0034] In one embodiment, provided herein is a composition comprising the engineered protein described in any one of the embodiments herein conjugated to an antibody.

[0035] [Invention 1001] (i) Any one of amino acids 19 to 489 of SEQ ID NOs: 1, 2, and 4 to 7, or (ii) an engineered protein comprising the extracellular domain of the metapneumovirus (MPV) F protein having at least 90% identity to amino acids 19 to 484 of SEQ ID NO: 3, wherein the engineered protein comprises at least one mutation with respect to any one of the sequences of SEQ ID NOs: 1 to 7, and the at least one mutation comprises a substitution at a position corresponding to K166, N342, A / D185, K188, T49, V262, H435, E26, G439, N46, L158, A161, L50, V162, E51, R163, V104, N457, L110, N322, A113, D336, A116, A338, A140, A147, S291, S443, S293, S444, S355, V442, T365, V463, S22, G53, V169, E305, L302, V47, A159, T127, N153, G121, I / F258, G106, A107, T160, I128, A190, V118, Q426, L165, V191, S149, I137, V / I122, S192, T317, L105, L134, A117, S347, G261, I268, S470, L473, S265, L460, F48, Q455, V231, A374, I217, S376, G366, S194, L219, A344, A86, T114, V148, D461, L66, L73, N145, Q195, E453, and / or H368. [Inventive item 1002] The engineered protein of Inventive item 1001, comprising a proline substitution corresponding to A / D185P. [Inventive item 1003] The engineered protein of Inventive item 1001, comprising a substitution corresponding to RQSR (residues 99 to 102 of any one of SEQ ID NOs: 1 to 7) to RRRR (SEQ ID NO: 10). [Inventive item 1004] The engineered protein of Inventive item 1001, comprising a proline substitution corresponding to A / D185P and a substitution corresponding to RQSR (residues 99 to 102 of any one of SEQ ID NOs: 1 to 7) to RRRR (SEQ ID NO: 10). [Inventive item 1005] An engineered protein of the present invention 1001, comprising a substitution from any one of residues 87 to 104 of SEQ ID NO: 1 to 7 to GGGGSGGGGSR (SEQ ID NO: 8). [The present invention 1006] Engineered disulfide bonds comprising pairs of cysteine substitutions corresponding to E26C and G439C, N46C and L158C, T49C and A161C, L50C and V162C, E51C and R163C, E51C and K166C, V104C and N457C, L110C and N322C, A113C and D336C, A116C and A338C, A140C and A147C, S291C and S443C, S293C and S443C, S293C and S444C, S355C and V442C, T365C and V463C, S22C and H435C, G53C and K166C, G53C and V169C, E305C and N457C, S291C and L302C, V47C and A159C, T127C and N153C, G121C and I / F258C, F48C and T160C, and / or T365C and Q455C An engineered protein of any one of the present inventions 1001 to 1005, comprising the same. [The present invention 1007] Engineered disulfide bonds comprising pairs of cysteine substitutions corresponding to A116C and A338C, T365C and V463C, T127C and N153C, T365C and Q455C, V104C and N457C, L110C and N322C, or A140C and A147C An engineered protein of the present invention 1006, comprising the same. [The present invention 1008] Engineered disulfide bonds comprising pairs of cysteine substitutions corresponding to A140C and A147C An engineered protein of the present invention 1007, comprising the same. [The present invention 1009] Engineered disulfide bonds comprising pairs of cysteine substitutions corresponding to V104C and N457C An engineered protein of the present invention 1007, comprising the same. [The present invention 1010] Engineered disulfide bonds comprising pairs of cysteine substitutions corresponding to L110C and N322C An engineered protein of the present invention 1007, comprising the same. [The present invention 1011] Engineered disulfide bonds comprising pairs of cysteine substitutions corresponding to T365C and V463C An engineered protein of the present invention 1007, comprising the same. [The present invention 1012] An engineered protein of the present invention 1011, further comprising a substitution at a position corresponding to L219 and / or V231. [The present invention 1013] An engineered protein of the present invention 1011, further comprising a substitution corresponding to L219K and / or V231I. [The present invention 1014] An engineered protein of any one of the present inventions 1011 to 1013, further comprising a pair of cysteine substitutions corresponding to T127C and N153C. [The present invention 1015] An engineered protein of any one of the present inventions 1011 to 1013, further comprising a pair of cysteine substitutions corresponding to L110C and N322C. [The present invention 1016] An engineered protein of any one of the present inventions 1011 to 1015, further comprising a pair of cysteine substitutions corresponding to A140C and A147C. [The present invention 1017] A further substitution corresponding to G366S, an engineered protein of any one of the present inventions 1011 to 1016. [The present invention 1018] An engineered protein of the present invention 1011, further comprising a substitution at a position corresponding to Q426, T49, L187, L473, and / or S347. [The present invention 1019] An engineered protein of the present invention 1011, further comprising a substitution at a position corresponding to Q426, T49, L187, L473, and / or S347. [The present invention 1020] The engineered protein of the present invention 1011 further comprising a substitution corresponding to Q426W, T49E, L187F, L473F, and / or S347Q. [The present invention 1021] An engineered disulfide bond comprising a pair of cysteine substitutions corresponding to A116C and A338C The engineered protein of the present invention 1007 comprising the same. [The present invention 1022] An engineered disulfide bond comprising a pair of cysteine substitutions corresponding to T365C and V463C The engineered protein of the present invention 1007 comprising the same. [The present invention 1023] An engineered disulfide bond comprising a pair of cysteine substitutions corresponding to T127C and N153C The engineered protein of the present invention 1007 comprising the same. [The present invention 1024] An engineered disulfide bond comprising a pair of cysteine substitutions corresponding to T365C and Q455C The engineered protein of the present invention 1007 comprising the same. [The present invention 1025] The engineered protein of the present invention 1007 further comprising at least one additional engineered disulfide bond. [The present invention 1026] The engineered protein of any one of the present inventions 1001 - 1005 comprising a cavity filling substitution at a position corresponding to G106, A107, T160, L158, I128, A190, V118, Q426, L165, V191, T160, S149, I137, S149, V169, N46, T49, V / I122, S192, T317, V162, L105, L134, A117, S347, V47, G261, I268, S470, V231, A374, I217, and / or S355. [The present invention 1027] The engineered protein of the present invention 1026 comprising a cavity filling substitution at a position corresponding to L105, V118, I137, S149, L158, L165, or Q426. [The present invention 1028] An engineered protein of the present invention 1027, comprising a substitution corresponding to L105I or L105W. [The present invention 1029] An engineered protein of the present invention 1027, comprising a substitution corresponding to L158W. [The present invention 1030] An engineered protein of the present invention 1027, comprising a substitution corresponding to V118F or V118M. [The present invention 1031] An engineered protein of the present invention 1027, comprising a substitution corresponding to Q426W. [The present invention 1032] An engineered protein of the present invention 1027, comprising a substitution corresponding to L165F. [The present invention 1033] An engineered protein of the present invention 1027, comprising a substitution corresponding to S149V or S149I. [The present invention 1034] An engineered protein of the present invention 1033, further comprising a substitution at the position corresponding to I137. [The present invention 1035] An engineered protein of the present invention 1034, comprising a substitution corresponding to I137L. [The present invention 1036] An engineered protein of the present invention 1026, comprising a cavity-filling substitution selected from the group consisting of G106W, A107F, T160M, L158W, I128F, A190M, V118F, V118M, Q426W, L165F, V191I, T160V, S149V, I137L, S149I, V169I, N46V, T49I, V / I122L, S192L, T317L, V162F, V162W, L105I, L105F, L105W, L134I, A117M, S347M, S347K, S347Q, V47M, G261M, I268M, S470Y, V231I, A374V, I217V, and / or S355F. [The present invention 1037] An engineered protein of any one of the present invention 1001 to 1005, comprising a proline substitution selected from the group consisting of A86P, A107P, A113P, T114P, V148P, S443P, D461P, L130P, L141P, K142P, E146P, L151P, N153P, V162P, A / D185P, D186P, L187P, K188P, N342P, and A344P. [The present invention 1038] An engineered protein of any one of the present invention 1001 to 1005, comprising a substitution at a position corresponding to S376, G366, and / or S194. [The present invention 1039] An engineered protein of the present invention 1038, comprising a substitution corresponding to S376T, G366S, and / or S194Q. [The present invention 1040] An engineered protein of any one of the present invention 1001 to 1005, comprising a substitution at a position corresponding to K166. [The present invention 1041] An engineered protein of the present invention 1040, comprising a substitution corresponding to K166E. [The present invention 1042] An engineered protein of any one of the present invention 1001 to 1005, comprising a substitution for adjusting pH sensitivity at a position corresponding to H435. [The present invention 1043] An engineered protein of the present invention 1042, comprising a substitution corresponding to H435E, H435D, or H435N. [The present invention 1044] An engineered protein of any one of the present invention 1001 to 1005, comprising an electrostatic interaction substitution at a position corresponding to L66, L73, N145, Q195, E453, L66, K188, H368, D461, T49, and / or V262. [The present invention 1045] An engineered protein of the present invention 1044, comprising a substitution corresponding to L66N, L73E, N145E, Q195K, E453Q, L66D, K188R, H368R, D461E, T49E, and / or V262D. [The present invention 1046] An engineered protein of any one of the present inventions 1001 to 1005, comprising substitutions corresponding to L110C, T127C, A140C, A147C, N153C, L219K, V231I, N322C, T365C, E453Q, and / or V463C. [The present invention 1047] An engineered protein of any one of the present inventions 1001 to 1005, comprising substitutions corresponding to T127C, N153C, A185P, T365C, V463C, L219K, and V231I. [The present invention 1048] An engineered protein of any one of the present inventions 1001 to 1005, comprising substitutions corresponding to T127C, N153C, A185P, T365C, V463C, L219K, V231I, and the substitution of RQSR (residues 99 to 102 of any one of SEQ ID NOs: 1 to 7) with RRRR (SEQ ID NO: 10). [The present invention 1049] An engineered protein of any one of the present inventions 1001 to 1005, comprising substitutions corresponding to T127C, N153C, T365C, V463C, L219K, and V231I. [The present invention 1050] An engineered protein of any one of the present inventions 1001 to 1005, comprising substitutions corresponding to L110C, T127C, A140C, A147C, N153C, A185P, L219K, V231I, N322C, T365C, N368H, E453Q, and V463C. [The present invention 1051] An engineered protein of any one of the present inventions 1001 to 1005, comprising substitutions corresponding to L110C, T127C, A140C, A147C, N153C, A185P, L219K, V231I, N322C, T365C, N368H, E453Q, V463C, and the substitution of RQSR (residues 99 to 102 of any one of SEQ ID NOs: 1 to 7) with RRRR (SEQ ID NO: 10). [The present invention 1052] An engineered protein of any of the present inventions 1001 to 1005, comprising substitutions corresponding to L110C, T127C, A140C, A147C, N153C, L219K, V231I, N322C, T365C, N368H, E453Q, and V463C. [The present invention 1053] An engineered protein of any of the present inventions 1006 to 1052, comprising a substitution corresponding to GGGGSGGGGSR (SEQ ID NO: 8) from residue 87 to 104 of any one of SEQ ID NOs: 1 to 7. [The present invention 1054] An engineered protein of any of the present inventions 1001 to 1053, comprising a combination of at least one engineered disulfide bond, at least one cavity-filling substitution, and at least one proline substitution. [The present invention 1055] An engineered protein of any of the present inventions 1001 to 1054, comprising a polypeptide sequence that is at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 14 or 16. [The present invention 1056] An engineered protein of the present inventions 1001 to 1054, comprising a polypeptide sequence that is at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 15 or 17. [The present invention 1057] An engineered protein of any of the present inventions 1001 to 1056, having at least 95% identity to the amino acid sequence of SEQ ID NOs: 1 to 3. [The present invention 1058] An engineered protein of any of the present inventions 1001 to 1056, comprising an engineered hMPV F protein extracellular domain having 95% identity to SEQ ID NO: 3. [The present invention 1059] An engineered protein of any of the present inventions 1001 to 1058, wherein the protein is fused or conjugated to a trimerization domain. [The present invention 1060] The engineered protein of the present invention 1059, wherein the protein is fused to a trimerization domain. [The present invention 1061] The engineered protein of the present invention 1060, wherein the trimerization domain comprises the trimerization domain of T4 fibritin. [The present invention 1062] The engineered protein of any one of the present inventions 1001 to 1058, wherein the protein is fused or conjugated to a transmembrane domain. [The present invention 1063] The engineered protein of the present invention 1062, wherein the protein is fused to a transmembrane domain. [The present invention 1064] The engineered protein of the present invention 1062, wherein the transmembrane domain comprises the transmembrane domain of the metapneumovirus (MPV) F protein. [The present invention 1065] The engineered protein of any one of the present inventions 1001 to 1062, comprising an N-terminal signal sequence. [The present invention 1066] The engineered protein of the present invention 1065, wherein the N-terminal signal sequence is MSWKVMIIISLLITPQHG (SEQ ID NO: 11). [The present invention 1067] A trimer of an engineered metapneumovirus (MPV) F protein comprising at least one subunit of any one of the present inventions 1001 to 1066. [The present invention 1068] The engineered trimer of the present invention 1067, wherein the trimer is stabilized in the pre-fusion conformation as compared to the trimer of the wild-type metapneumovirus (MPV) F subunit. [The present invention 1069] The engineered trimer of the present invention 1067, wherein the trimer comprises at least one engineered disulfide bond between subunits. [The present invention 1070] The engineered trimer of the present invention 1069, wherein at least one engineered disulfide bond between subunits is selected from S316C and D421C. [The present invention 1071] A pharmaceutically acceptable carrier, and (i) Any of the engineered proteins of the present invention 1001 to 1066, or (ii) any of the engineered trimers of the present invention 1067 to 1070 A pharmaceutical composition comprising the same. [The present invention 1072] The composition of the present invention 1071, further comprising an adjuvant. [The present invention 1073] A nucleic acid molecule comprising a nucleotide sequence encoding the amino acid sequence of any of the engineered proteins of the present invention 1001 to 1066. [The present invention 1074] The nucleic acid of the present invention 1073, wherein the nucleic acid comprises a DNA expression vector. [The present invention 1075] The nucleic acid of the present invention 1073, wherein the nucleic acid comprises mRNA. [The present invention 1076] A method for preventing metapneumovirus (MPV) infection or a disease associated with MPV infection in a subject, the method comprising administering to the subject an effective amount of any of the pharmaceutical compositions of the present invention 1071 to 1072 or any of the nucleic acid molecules of the present invention 1073 to 1075. [The present invention 1077] Any of the pharmaceutical compositions of the present invention 1071 to 1072 or any of the nucleic acid molecules of the present invention 1073 to 1075 for use in the treatment or prevention of metapneumovirus (MPV) infection or a disease associated with MPV infection in a subject. [The present invention 1078] Use of any of the pharmaceutical compositions of the present invention 1071 to 1072 or any of the nucleic acid molecules of the present invention 1073 to 1075 in the manufacture of a medicament for the treatment or prevention of metapneumovirus (MPV) infection or a disease associated with MPV infection. [The present invention 1079] A composition comprising an engineered protein of any one of 1001-1066 of the present invention or an engineered trimer of any one of 1067-1070 of the present invention, bound to an antibody. Other objects, features, and advantages of the present invention will become apparent from the following detailed description. However, while this detailed description and specific examples represent preferred embodiments of the present invention, it should be understood that they are given by way of illustration only, since various changes and modifications within the spirit and scope of the present invention will become apparent to those skilled in the art from this detailed description.

Brief Description of the Drawings

[0036] The following drawings form a part of this specification and are included to further illustrate certain aspects of the present invention. The present invention can be better understood by reference to one or more of these drawings in combination with the detailed description of specific embodiments presented herein.

[0037]

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Mode for Carrying Out the Invention

[0038] Detailed Description The human metapneumovirus (hMPV) fusion (F) protein is essential for virus entry and is an important target for neutralizing antibodies and vaccine development. The prefusion conformation is thought to be an optimal vaccine antigen, but previously described expression of the prefusion F protein has been insufficient and poorly stabilized. In the present invention, the structure of hMPV F was used to guide design and characterize engineered hMPV F proteins. In some embodiments, the engineered hMPV F proteins described in the embodiments are stabilized in the conformation that exists prior to membrane fusion. Such engineered proteins can be used, for example, to stimulate an immune response specific for the anti-hMPV F protein. In further embodiments, the engineered F protein can be used to detect F protein-binding antibodies in a sample. Thus, the engineered proteins provided herein enable a more effective method for vaccination against hMPV and, at the same time, validate a new assay method for detecting anti-hMPV F protein antibodies, for example, in a biological sample.

[0039] I. Aspects of the Disclosure Similar to other class I fusion proteins, the hMPV F protein is readily induced by host factors and transitions from a metastable prefusion state to a highly stable postfusion state. The use of prefusion-stabilizing proteins such as DS-Cav1 as vaccinogens has been shown to elicit high neutralizing titers in animal models compared to the use of postfusion proteins. This provided the idea for the use of the prefusion structure of hMPV F (PDB ID: 5WB0) as a guide for strategically introducing mutations one at a time specifically into regions that undergo substantial conformational changes during the pre-to-post transition. Similar to recent successes in engineering the SARS-CoV-2 spike, multiple proline substitutions increased protein expression levels while maintaining F in its prefusion conformation. The role of A107P in the fusion peptide is quite similar to that of the F817P substitution from HexaPro (Hsieh et al., 2020), likely by imposing rigidity on the fusion peptide and also capping the helix in its prefusion conformation. Interestingly, replacing Ala with Phe, the equivalent residue on RSV F, resulted in a slight decrease in protein expression levels compared to the basic construct (Table 1). D461P in the HRB may also perform the same function as A107P or A113P, which were the most effective proline substitutions within the fusion peptide. In antigenic site V, there are three proline substitutions that lead to an increase in protein yield. Considering that the side chains of these residues are partially exposed on the surface of the trimer, they are considered to be suboptimal for vaccine antigens.

[0040] The implementation of inter-protomer or inter-subunit salt bridges tends to efficiently retain the trimeric viral protein in a relatively compact conformation in a more effective manner than intra-protomer salt bridges. For example, the K588E mutation from gp41 electrostatically interacts with K62 or K492 from gp120, which is advantageous for the HIV-1 Env to remain in the pre-fusion closed conformation (Rutten et al., 2018). Among the present designs, the L219K substitution from the F1 subunit may form a salt bridge with either E80 from the F2 subunit or D209 from the F1 subunit. The 73E substitution from the F2 subunit may also form a salt bridge with R198 from the neighboring F1 subunit. Both variants enhance expression and have a longer retention time in SEC, which implicitly indicates a similar role of salt bridge design for class I fusion proteins. On the other hand, the intra-protomer salt bridge design such as the variant N145E abolished the expression of hMPV F (Table 1). Similarly, this type of salt bridge was not very effective for the stabilization of SARS-CoV-2 spike. Reduction of repulsion caused by charge clusters at the protomer interface is another approach to prevent the opening of the trimer. The charge cluster (E453 / D454) was found in the HRB region proximal to the base of the trimer. Therefore, replacing Glu453 with the equivalent Gln may reduce the charge repulsion caused by the negatively charged cluster. Similar to the E487Q substitution from RSV F or the K588F substitution from Ebola GP (McLellan et al., 2013, Rutten et al., 2020), this variant made hMPV F into a more compact trimer and retained the native quaternary structure as revealed by the SEC elution profile (Figure 2B).

[0041] The cavity filling approach was also very effective in stabilizing the loosely aggregated viral protein in its pre-fusion conformation. For example, the S190F substitution in the Cav1 variant successfully filled the cavity between site V and site II, and the V207L substitution successfully filled the pocket between sites Φ with only a single CH2 addition. V231I, one of the variants showing the highest expression, is surprisingly located in domain IIIb (site II), a region that undergoes no conformational change during the pre- to post-fusion transition. The other two substitutions that enhanced expression (S149I, I137L) are both located in domain IIIa (site V) and appear to gather together relative to each other to stabilize the highly flexible α2 and β3.

[0042] Among the strategies used to stabilize hMPV F, the introduction of disulfide bonds probably yields the highest success rate. The L110C / N322C substitution was designed to trap the fusion peptide within the central cavity, and the T365C / V463C substitution was designed to lock the HRB in the membrane-proximal region. By fixing the fusion peptide or HRB in a region that remains stationary during the pre- to post-fusion transition, the F protein is retained in its pre-fusion conformation and its thermal stability is significantly improved. This approach has been successfully used for several class I viral fusion proteins (McLellan et al., 2013, Stewart-Jones et al., 2018, Sanders et al., 2013). This design evokes the DS variant for RSV F and SOSIP for HIV-1 Env. In contrast, the T127C / N153C or A140C / A147C substitutions represent different types of disulfide design strategies. These disulfide bonds are placed in regions that undergo conformational changes (e.g., domain IIIa (site V)), but they appear to stabilize the pre-fusion state by preventing the refolding of HRA near the central helix. This is similar to the Q162C / L168C substitution used for the F protein of influenza virus 3 (Stewart-Jones et al., 2018), suggesting that this type of disulfide design could be a general approach for stabilizing class I viral fusion proteins. They can prevent the refolding of HRA and potentially force it to refold into its pre-fusion conformation. Similar disulfide designs that limit the local flexibility of the secondary structure in regions that undergo conformational changes during the pre- to post-fusion transition have also been successfully applied to SARS-CoV-2 spike. Interestingly, the V104C / N457C substitution blocks furin cleavage of F and yields a single species of F0 in SDS-PAGE. The furin cleavage site proximal to the fusion peptide may be buried in the central cavity due to the disulfide design. This variant could be practical for protease-free production of vaccine antigens.

[0043] Combining multiple beneficial modifications to increase protein expression levels and stability has proven to be an effective strategy for producing optimized prefusion antigens (Joyce et al., 2016, Krarup et al., 2015, McLellan et al., 2013, Rutten et al., 2020, Rutten et al., 2018, Jiachen et al., 2021, Hsieh et al., 2020). In the present invention, by combining multiple beneficial modifications, DS-CavEs2, one of the best constructs, including the design of disulfide bonds, cavity filling, and electrostatic stabilization, was obtained. DS-CavEs2 has 10-fold higher protein expression levels, enhanced thermal stability, and retains the prefusion epitope even after heat stress and long-term storage at 4°C. Two disulfide substitutions at site V, cavity filling substitutions at site II, and the introduction of another disulfide bond proximal to the fusion peptide did not disrupt the three-dimensional structure of the membrane-distal half of hMPV F, which has the most potent neutralizing epitope inside RSV F (Figure 8A) (Graham et al., 2017, Gilman et al., 2016). In contrast, the T365C / V463C substitution changed the relative position of the α10 helix. However, this membrane-proximal region is unlikely to be immunogenic, and no neutralizing antibodies targeting this region in RSV F have been discovered.

[0044] Another version of DS-CavEs2 in which the furin site is replaced with a flexible glycine-serine (GS) linker or polyglycine linker generates a single-chain form of the prefusion F trimer. In some embodiments, the linker has the sequence GGSGGS (SEQ ID NO: 12) or GGGGGG (SEQ ID NO: 13). Considering that no furin protease is required to recombinantly produce this construct, this design may be more cost-effective for the industrial production of vaccine antigens.

[0045] Notably, both pre-fusion stabilized F constructs crystallized as monomers even when complexed with MPE8, which recognizes epitopes spanning adjacent protomers. Other groups have also crystallized monomeric hMPV F bound to antibodies that recognize different antigenic sites (Huang et al., 2020; Wen et al., 2012). Considering that visualization of trimeric F particles by nsEM was possible (Figure 8B), these data suggest that the hMPV F trimer is in equilibrium with dissociated monomers even when fused to a trimerization motif. This is consistent with recent results demonstrating that some class I viral fusion proteins undergo trimer opening or "breathing," and native antibodies that bind to the trimer interface of influenza HA and hMPV F have been isolated (Bangaru et al., 2019; Watanabe et al., 2019; Gilman et al., 2019).

[0046] Pre-fusion stabilized class I virus fusion proteins are known to elicit high neutralizing antibody titers that are often an order of magnitude higher than those induced by post-fusion antigens in animals and humans (Crank et al., 2019, McLellan et al., 2013, Stewart-Jones et al., 2018). Previous studies showed little difference in the immunogenicity of pre-fusion and post-fusion hMPV F proteins (Battles et al., 2017), but those studies were performed using post-fusion F proteins with an antigen dose of 10 μg and some contamination with pre-fusion-like proteins that had not yet adopted the post-fusion conformation, which is now known. Pre-fusion stabilized hMPV F antigens are expected to elicit higher neutralizing antibody titers in mice than post-fusion F proteins. These results are more consistent with other immunogenicity studies of pre-fusion stabilized viral proteins than previous hMPV F studies (Crank et al., 2019, van den Hoogen et al., 2002, Stewart-Jones et al., 2018). The stabilized proteins described herein should facilitate the isolation of potent and broadly reactive monoclonal antibodies that could accelerate the development of hMPV F vaccine candidates and be useful for the passive prophylaxis of high-risk cohorts.

[0047] II. Definitions It should be understood that both the foregoing general description and the following detailed description are exemplary and explanatory only, and do not limit the claimed invention. In this application, the use of the singular form includes the plural form unless otherwise specifically defined. In this application, the use of "or" means "and / or" unless otherwise specified. Further, the term "including" and the use of other forms such as "includes" and "included" are not limiting. Also, terms such as "element" or "component" include both elements and components that include one unit and elements and components that include more than one subunit, unless otherwise specifically defined. Also, the use of the term "portion" may include a part or the whole of a moiety.

[0048] As used herein, the notation "a" or "an" may mean one or more. As used herein, when used in conjunction with the phrase "comprising" in a claim(s), the word "a" or "an" may mean one or more than one.

[0049] The use of the term "or" in the claims is used to mean "and / or" as used herein, unless explicitly indicated to refer only to alternatives or the alternatives are mutually exclusive, even if the disclosure supports a definition that refers only to alternatives and "and / or". As used herein, "another" may mean at least a second or later.

[0050] As used herein when referring to measurable values such as amounts, durations of time, etc., the term "about" is intended to encompass variations of up to ±10% from the stated value. Unless otherwise indicated, all numbers expressing amounts of ingredients, properties such as molecular weights, reaction conditions, etc. used in the specification and claims are to be understood as being modified in all instances by the term "about". Accordingly, unless indicated to the contrary, the numerical parameters set forth in the following specification and the appended claims are approximations that may vary depending upon the desired properties sought to be obtained by the disclosed invention. At the very least, and not as an attempt to limit the application of the doctrine of equivalents to the claims, each numerical parameter should be construed in light of the reported number of significant digits and by applying ordinary rounding techniques. Notwithstanding that the numerical ranges and parameters setting forth the broad scope of the invention are approximations, the numerical values set forth in the specific examples are reported as precisely as possible. However, any numerical value inherently contains certain errors necessarily resulting from the standard deviation found in their respective test measurements.

[0051] As used herein, the term "essentially free of" with respect to a specified component means that none of the specified components are intentionally formulated into the composition and / or are present only as impurities or in trace amounts. Accordingly, the total amount of the specified component resulting from any unintentional impurities in the composition is well below 0.05%, preferably below 0.01%. Most preferred are compositions in which no amount of the specified component can be detected by standard analytical methods.

[0052] The term "antibody" refers to intact immunoglobulins of any isotype, or fragments thereof that can compete with intact antibodies for specific binding to a target antigen, and includes, for example, chimeric, humanized, fully human, and bispecific antibodies. An "antibody" is a species of antigen-binding protein. Intact antibodies generally include at least two full-length heavy chains and two full-length light chains, but in some cases may include fewer chains, such as antibodies that naturally occur in camels and contain only heavy chains. Antibodies can be derived from a single source only or can be "chimeric," i.e., different portions of the antibody can be derived from two different antibodies, as further described below. Antigen-binding proteins, antibodies, or binding fragments can be produced in hybridomas, by recombinant DNA techniques, or by enzymatic or chemical cleavage of intact antibodies. Unless otherwise indicated, the term "antibody" includes, in addition to antibodies containing two full-length heavy chains and two full-length light chains, derivatives, variants, fragments, and mutant proteins thereof, examples of which are described below. Further, unless explicitly excluded, antibodies include monoclonal antibodies, bispecific antibodies, minibodies, domain antibodies, synthetic antibodies (sometimes referred to herein as "antibody mimetics"), chimeric antibodies, humanized antibodies, human antibodies, antibody fusions (sometimes referred to herein as "antibody conjugates"), and fragments thereof, respectively. In some embodiments, the term also encompasses peptibodies.

[0053] The structural unit of a native antibody includes a tetramer. Each such tetramer typically consists of a pair of two identical polypeptide chains, each pair having one full-length "light" chain (in certain embodiments, about 25 kDa) and one full-length "heavy" chain (in certain embodiments, about 50 - 70 kDa). The amino-terminal portion of each chain typically includes a variable region of about 100 - 110 or more amino acids that typically bears antigen recognition. The carboxy-terminal portion of each chain typically defines a constant region that can bear effector functions. Human light chains are typically classified as kappa light chains and lambda light chains. Heavy chains are typically classified as mu, delta, gamma, alpha, or epsilon, which define the antibody isotypes as IgM, IgD, IgG, IgA, and IgE, respectively. IgG has several subclasses including, but not limited to, IgG1, IgG2, IgG3, and IgG4. IgM has subclasses including, but not limited to, IgM1 and IgM2. IgA is similarly subdivided into subclasses including, but not limited to, IgA1 and IgA2. Within the full-length light and heavy chains, typically, the variable and constant regions are linked by a "J" region of about 12 or more amino acids, and the heavy chain also typically includes a "D" region of about 10 more amino acids. See, for example, Fundamental Immunology, Ch. 7 (Paul, W., ed., 2nd ed. Raven Press, N.Y. (1989)) (which is hereby incorporated by reference in its entirety for all purposes). The variable regions of each light chain / heavy chain pair typically form the antigen-binding site.

[0054] The term "variable region" or "variable domain" refers to a portion of the light and / or heavy chains of an antibody that typically includes approximately 120 - 130 amino acids at the amino terminus in the heavy chain and about 100 - 110 amino-terminal amino acids in the light chain. In certain embodiments, the variable regions of different antibodies can have significantly different amino acid sequences even among antibodies of the same species. The variable region of an antibody typically determines the specificity of that antibody for its target.

[0055] The variable regions typically exhibit the same general structure in which a relatively conserved framework region (FR) is linked by three hypervariable regions (also called complementarity-determining regions or CDRs). The CDRs from each pair of two chains are typically aligned by the framework regions, which can enable binding to a specific epitope. From the N-terminus to the C-terminus, both the light chain variable region and the heavy chain variable region typically include domains FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4. The amino acid assignments to each domain typically follow the definitions of Kabat Sequences of Proteins of Immunological Interest (National Institutes of Health, Bethesda, Md. (1987 and 1991)), Chothia & Lesk J. Mol. Biol., 196:901-917 (1987), or Chothia et al., Nature, 342:878-883 (1989).

[0056] In certain embodiments, the antibody heavy chain binds to an antigen in the absence of the antibody light chain. In certain embodiments, the antibody light chain binds to an antigen in the absence of the antibody heavy chain. In certain embodiments, the antibody binding region binds to an antigen in the absence of the antibody light chain. In certain embodiments, the antibody binding region binds to an antigen in the absence of the antibody heavy chain. In certain embodiments, an individual variable region specifically binds to an antigen in the absence of other variable regions.

[0057] In certain embodiments, the definitive delineation of the CDRs and the identification of the residues that constitute the binding site of the antibody are accomplished by elucidating the structure of the antibody and / or the structure of the antibody-ligand complex. In certain embodiments, it can be accomplished by any of a variety of techniques known to those of skill in the art, such as X-ray crystallographic analysis. In certain embodiments, various analytical methods can be used to identify or approximately predict the CDR regions. Examples of such methods include, but are not limited to, the Kabat definition, the Chothia definition, the AbM definition, and the contact definition.

[0058] The Kabat definition is a standard for numbering residues in antibodies and is typically used to identify CDR regions. See, for example, Johnson & Wu, Nucleic Acids Res., 28:214-8 (2000). The Chothia definition is similar to the Kabat definition, but the Chothia definition takes into account the position of certain structural loop regions. See, for example, Chothia et al., J. Mol. Biol., 196:901-17 (1986), Chothia et al., Nature, 342:877-83 (1989). The AbM definition uses an integrated set of computer programs manufactured by Oxford Molecular Group to model antibody structures. See, for example, Martin et al., Proc Natl Acad Sci (USA), 86:9268-9272 (1989), “AbM TM , A Computer Program for Modeling Variable Regions of Antibodies,” Oxford, UK; Oxford Molecular, Ltd. The AbM definition uses a combination of a knowledge database and ab initio methods such as those described in Samudrala et al., “Ab Initio Protein Structure Prediction Using a Combined Hierarchical Approach,” in PROTEINS, Structure, Function and Genetics Suppl., 3:194-198 (1999) to model the tertiary structure of an antibody from its primary sequence. The contact definition is based on the analysis of available complex crystal structures. See, for example, MacCallum et al., J. Mol. Biol., 5:732-45 (1996).

[0059] Customarily, the CDR regions of the heavy chain are typically designated as H1, H2, and H3, and are sequentially numbered in the direction from the amino terminus to the carboxy terminus. The CDR regions of the light chain are typically designated as L1, L2, and L3, and are sequentially numbered in the direction from the amino terminus to the carboxy terminus.

[0060] The term "light chain" includes the full-length light chain and fragments thereof having a variable region sequence sufficient to confer binding specificity. The full-length light chain includes the variable region domain VL and the constant region domain CL. The variable region domain of the light chain is at the amino terminus of the polypeptide. Light chains include kappa chains and lambda chains.

[0061] The term "heavy chain" includes the full-length heavy chain and fragments thereof having a variable region sequence sufficient to confer binding specificity. The full-length heavy chain includes the variable region domain VH, as well as three constant region domains CH1, CH2, and CH3. The VH domain is at the amino terminus of the polypeptide, the CH domains are at the carboxyl terminus, with CH3 being closest to the carboxy terminus of the polypeptide. Heavy chains can be of any isotype, including IgG (including IgG1, IgG2, IgG3, and IgG4 subtypes), IgA (including IgA1 and IgA2 subtypes), IgM, and IgE.

[0062] Bispecific or bifunctional antibodies are typically artificial hybrid antibodies having two different heavy chain / light chain pairs and two different binding sites. Bispecific antibodies can be produced by a variety of methods including, but not limited to, fusion of hybridomas or ligation of Fab’ fragments. See, for example, Songsivilai et al., Clin. Exp. Immunol., 79:315-321 (1990), Kostelny et al., J. Immunol., 148:1547-1553 (1992).

[0063] The term "antigen" refers to a substance that can induce an adaptive immune response. Specifically, an antigen is a substance that functions as a target for the receptors of the adaptive immune response. Typically, an antigen is a molecule that binds to an antigen-specific receptor but cannot itself induce an immune response in the body. Antigens are usually proteins and polysaccharides, and less frequently lipids. As used herein, the term "antigen" also includes immunogens and haptens.

[0064] The "Fc" region contains two heavy chain fragments that include the CH1 and CH2 domains of the antibody. These two heavy chain fragments are held together by two or more disulfide bonds and hydrophobic interactions of the CH3 domain.

[0065] The "Fv region" contains the variable regions from both the heavy and light chains but lacks the constant regions.

[0066] An antibody that "specifically binds to" or is "specific for" a particular polypeptide or an epitope on a particular polypeptide is an antibody that binds to the particular polypeptide or the epitope on the particular polypeptide without substantially binding to any other polypeptide or polypeptide epitope. For example, an hMPV F protein-specific antibody of the present invention is specific for the hMPV F protein. In some embodiments, an antibody that binds to the hMPV F protein has a dissociation constant (Kd) of ≤ 100 nM, ≤ 10 nM, ≤ 1 nM, ≤ 0.1 nM, ≤ 0.01 nM, or ≤ 0.001 nM (e.g., ≤ 10-8 M, e.g., 10-8 M to 10-13 M, e.g., 10-9 M to 10-13 M).

[0067] As used in the context of antigen-binding proteins (e.g., antibodies or antigen-binding fragments thereof) that compete for the same epitope, the term "compete" means that the antigen-binding protein being tested (e.g., an antibody or antigen-binding fragment thereof) inhibits or blocks (e.g., reduces) the specific binding of a reference antigen-binding protein (e.g., a ligand or reference antibody) to a common antigen (e.g., hMPV F or a fragment thereof), as determined by an assay. Whether one antigen-binding protein competes with another can be determined using a number of types of competitive binding assays, such as solid-phase direct or indirect radioimmunoassay (RIA), solid-phase direct or indirect enzyme immunoassay (EIA), sandwich competition assay (see, e.g., Stahli et al., 1983, Methods in Enzymology 9:242-253); solid-phase direct biotin-avidin EIA (see, e.g., Kirkland et al., 1986, J. Immunol. 137:3614-3619), solid-phase direct labeled assay, solid-phase direct labeled sandwich assay (see, e.g., Harlow and Lane, 1988, Antibodies, A Laboratory Manual, Cold Spring Harbor Press); solid-phase direct labeled RIA using I-125 labeling (see, e.g., Morel et al., 1988, Molec. Immunol. 25:7-15); solid-phase direct biotin-avidin EIA (see, e.g., Cheung, et al., 1990, Virology 176:546-552); and direct labeled RIA (Moldenhauer et al., 1990, Scand. J. Immunol. 32:77-82). Typically, such assays involve the use of a purified antigen bound to a solid surface or cell bearing any of these, an unlabeled test antigen-binding protein, and a labeled reference antigen-binding protein. Competitive inhibition is measured by determining the amount of label bound to the solid surface or cell in the presence of the test antigen-binding protein. Usually, the test antigen-binding protein is present in excess.Antigen-binding proteins identified by a competitive assay (competing antigen-binding proteins) include antigen-binding proteins that bind to the same epitope as the reference antigen-binding protein, and antigen-binding proteins that bind to adjacent epitopes that are proximal enough to cause steric hindrance to the epitope bound by the reference antigen-binding protein. Additional details regarding methods for determining competitive binding are provided in the Examples herein. Usually, when the competing antigen-binding protein is present in excess, it will inhibit (e.g., reduce) the specific binding of the reference antigen-binding protein to the common antigen by at least 40-45%, 45-50%, 50-55%, 55-60%, 60-65%, 65-70%, 70-75%, or 75% or more. In some cases, the binding is inhibited by at least 80-85%, 85-90%, 90-95%, 95-97%, or 97% or more.

[0068] As used herein, the term "epitope" refers to a specific group of atoms or amino acids on an antigen to which an antibody binds. An epitope can be either a linear epitope or a conformational epitope. A linear epitope is formed by a continuous amino acid sequence from an antigen and interacts with an antibody based on their primary structure. On the other hand, a conformational epitope is composed of discontinuous segments of an antigen's amino acid sequence and interacts with an antibody based on the antigen's 3D structure. Generally, an epitope is approximately 5 or 6 amino acids in length. When two antibodies exhibit competitive binding to an antigen, they may bind to the same epitope within the antigen.

[0069] The term "host cell" means a cell that has been transformed or can be transformed with a nucleic acid sequence and thereby expresses a gene of interest. This term includes the progeny of the parent cell, whether or not the progeny's form or genetic makeup is identical to that of the original parent cell, as long as the gene of interest is present.

[0070] The term "identity" refers to the relationship between the sequences of two or more polypeptide molecules or two or more nucleic acid molecules as determined by the alignment and comparison of the sequences. "Percent identity" means the percentage of residues that are identical between amino acids or nucleotides in the molecules being compared, and is calculated based on the size of the smallest molecule among the molecules being compared. For these calculations, gaps (if any) in the alignment are preferably treated by a particular mathematical model or computer program (i.e., an "algorithm"). Methods that can be used to calculate the identity of aligned nucleic acids or polypeptides include those described in Computational Molecular Biology, (Lesk, A.M., ed.), 1988, New York: Oxford University Press, Biocomputing Informatics and Genome Projects, (Smith, D.W., ed.), 1993, New York: Academic Press, Computer Analysis of Sequence Data, Part I, (Griffin, A.M., and Griffin, H.G., eds.), 1994, New Jersey: Humana Press, von Heinje, G., 1987, Sequence Analysis in Molecular Biology, New York: Academic Press, Sequence Analysis Primer, (Gribskov, M. and Devereux, J., eds.), 1991, New York: M. Stockton Press, and Carillo et al., 1988, SIAM J. Applied Math. 48:1073.

[0071] When calculating the percent identity, the sequences being compared are typically aligned to yield the maximum match between the sequences. An example of a computer program that can be used to determine the percent identity is the GCG program package, which includes GAP (Devereux et al., 1984, Nucl. Acid Res. 12:387, Genetics Computer Group, University of Wisconsin, Madison, Wis.). The computer algorithm GAP is used to align two polypeptides or polynucleotides whose percent sequence identity is to be determined. The sequences are aligned so that their corresponding amino acids or nucleotides are optimally matched (a "matched region" as determined by the algorithm). A gap opening penalty (which is calculated as 3× the average diagonal element, where the "average diagonal element" is the average of the diagonal elements of the comparison matrix being used and the "diagonal element" is the score or number assigned to each perfect match of amino acids by a particular comparison matrix) and a gap extension penalty (which is typically 1 / 10× the gap opening penalty), as well as a comparison matrix such as PAM 250 or BLOSUM 62 are used in conjunction with the algorithm. In certain embodiments, standard comparison matrices (see Dayhoff et al., 1978, Atlas of Protein Sequence and Structure 5:345-352 for the PAM 250 comparison matrix and Henikoff et al., 1992, Proc. Natl. Acad. Sci. U.S.A. 89:10915-10919 for the BLOSUM 62 comparison matrix) are also used by the algorithm.

[0072] Examples of parameters that can be used when determining the percent identity of polypeptide or nucleotide sequences using the GAP program can be found in Needleman et al., 1970, J. Mol. Biol. 48:443-453.

[0073] A particular alignment scheme for aligning two amino acid sequences may result in a match of only a short region of the two sequences, and this small alignment region may have a very high sequence identity even when there is no significant relationship between these two full-length sequences. Thus, the selected alignment method (the GAP program) can be adjusted, if so desired, to result in an alignment spanning at least 50 or some other number of contiguous amino acids of the target polypeptide.

[0074] As used herein, the term "linked" refers to an association through intramolecular interactions, such as covalent, metallic, and / or ionic bonds, or through intermolecular interactions, such as hydrogen bonds or non-covalent bonds.

[0075] The term "operably linked" refers to the arrangement of elements such that the components so described are configured to perform their normal functions. Thus, a given signal peptide operably linked to a polypeptide directs the secretion of the polypeptide from the cell. In the case of a promoter, a promoter operably linked to a coding sequence directs the expression of the coding sequence. A promoter or other regulatory element need not be contiguous with the coding sequence so long as they function to direct the expression of the coding sequence. For example, an intervening sequence that is untranslated but transcribed can be present between the promoter sequence and the coding sequence, and the promoter sequence can still be considered to be "operably linked" to the coding sequence.

[0076] The term "polynucleotide" or "nucleic acid" includes both single-stranded nucleotide polymers and double-stranded nucleotide polymers. The nucleotides that make up a polynucleotide can be ribonucleotides or deoxyribonucleotides, or modified forms of either type of nucleotide. Such modifications include base modifications such as bromouridine and inosine derivatives, ribose modifications such as 2',3'-dideoxyribose, and internucleotide linkage modifications such as phosphorothioate, phosphorodithioate, phosphorosenoate, phosphorodiselenoate, phosphoroanilothioate, phoshoraniladate, and phosphoramidate.

[0077] As used herein, the term "vector" refers to a nucleic acid molecule that is introduced into a host cell and thereby produces a transformed host cell. The vector may contain nucleic acid sequences that enable replication within the host cell, such as an origin of replication. The vector may also contain one or more therapeutic genes and / or selectable marker genes, as well as other genetic elements known in the art. The vector can transduce, transform, or infect a cell, thereby causing the cell to express nucleic acids and / or proteins other than those native to the cell. Optionally, the vector includes substances that assist in achieving entry of the nucleic acid into the cell, such as viral particles, liposomes, protein coatings, and the like.

[0078] The term "polypeptide" or "protein" means a macromolecule having the amino acid sequence of a native protein, i.e., a protein produced by native, non-recombinant cells, or it includes a molecule produced by genetic manipulation or recombinant cells that has the amino acid sequence of a native protein, or a molecule having a deletion, addition, and / or substitution of one or more amino acids of the native sequence. This term also includes amino acid polymers in which one or more amino acids are chemical analogs of the corresponding native amino acids and polymers. The terms "polypeptide" and "protein" expressly encompass an hMPV F protein-binding protein, an antibody, or a sequence having a deletion, addition, and / or substitution of one or more amino acids of an antigen-binding protein. The term "polypeptide fragment" refers to a polypeptide having an amino-terminal deletion, a carboxyl-terminal deletion, and / or an internal deletion as compared to a full-length native protein. Such fragments may also contain modified amino acids as compared to the native protein. In certain embodiments, the fragment is about 5 to 500 amino acids in length. For example, the fragment can be at least 5, 6, 8, 10, 14, 20, 50, 70, 100, 110, 150, 200, 250, 300, 350, 400, or 450 amino acids in length. Useful polypeptide fragments include immunologically functional fragments of antibodies, including binding domains. In the case of an hMPV F protein-binding antibody, useful fragments include, but are not limited to, CDR regions, variable domains of the heavy and / or light chains, a portion of an antibody chain containing two CDRs, or just its variable region.

[0079] The pharmaceutically acceptable carriers useful in the present invention are conventional. Remington’s Pharmaceutical Sciences, by E.W. Martin, Mack Publishing Co., Easton, PA, 15th Edition (1975) describes compositions and formulations suitable for the pharmaceutical delivery of the fusion proteins disclosed herein. Generally, the nature of the carrier will depend on the particular mode of administration being employed. For example, parenteral formulations usually include injectable solutions that contain pharmaceutically and physiologically acceptable fluids such as water, saline, balanced salt solutions, aqueous dextrose, glycerol, and the like, as a vehicle. In the case of solid compositions (e.g., powder, pill, tablet, or capsule forms), conventional non-toxic solid carriers can include, for example, pharmaceutical grade mannitol, lactose, starch, or magnesium stearate. In addition to biologically neutral carriers, the pharmaceutical compositions administered may contain minor amounts of non-toxic auxiliary substances such as wetting or emulsifying agents, preservatives, and pH buffering agents, such as sodium acetate or sorbitan monolaurate.

[0080] As used herein, the term “subject” refers to a human or any non-human animal (e.g., mouse, rat, rabbit, dog, cat, cow, pig, sheep, horse, or primate). Humans include both prenatal and postnatal forms. In many embodiments, the subject is a human. The subject can be a patient, which refers to a human who visits a healthcare provider for diagnosis or treatment of a disease. The term “subject” is used interchangeably herein with “individual” or “patient”. The subject may or may not be suffering from, or be susceptible to, a disease or disorder and may or may not exhibit symptoms of the disease or disorder.

[0081] As used herein, the terms “therapeutically effective amount” or “effective dosage” refer to the dosage or concentration of a drug that is effective in treating a disease or condition. For example, with respect to the use of the monoclonal antibodies or antigen-binding fragments thereof disclosed herein for treating viral infections.

[0082] As used herein, "treating" a condition or its "treatment" includes preventing or alleviating the condition, slowing the rate of onset or development of the condition, reducing the risk of developing the condition, preventing or delaying the onset of symptoms associated with the condition, reducing or eliminating symptoms associated with the condition, causing complete or partial regression of the condition, curing the condition, or some combination thereof.

[0083] III. hMPV F protein Human metapneumovirus (hMPV) is an enveloped, negative-strand virus of the family Pneumoviridae that was discovered in 2001 but had been circulating for at least half a century prior to its discovery. The hMPV fusion (F) protein is one of three surface glycoproteins encoded by the viral genome. As a class I fusogen, hMPV F is initially translated as a single polypeptide precursor (F0). Since it is initially non-functional, a proteolytic cleavage event is required to form the F1 and F2 subunits that are covalently linked by disulfide bonds. The new N-terminus of the F2 polypeptide contains a hydrophobic sequence that will be inserted into the host cell membrane during the process of ultimately fusing the viral and host cell membranes. At some point, either in transit or at the membrane surface, the F protein associates with itself to form a metastable trimer in the so-called pre-fusion conformation. The hMPV fusion protein is cleaved extracellularly by a trypsin-like protease. An unknown triggering event occurs, and in response, the F protein undergoes a dramatic conformational change in which, after extending the fusion peptide into the host cell membrane, it refolds back onto itself to form a six-helix bundle in the so-called post-fusion conformation. The energy difference between the extended intermediate and the post-fusion conformation provides the energy required for membrane fusion.

[0084] Historically, the structure of paramyxovirus fusion proteins has been classified into domains that generally distinguish the prefusion protein into head, neck, and stalk regions. Nomenclature assigning three general domains (DI, DII, and DIII) to the prefusion structure of additional constructs has continued. Since the Pneumoviridae family was formerly a subfamily of paramyxoviruses, they also retain this convention. The domains of hMPV are broadly classified into the same three domains, and two additional regions of heptad repeats are similarly defined. The heptad repeat A (HRA) is at the N-terminus of F1 located within DIIIa, and the heptad repeat B is C-terminal outside the defined domain region and prior to the transmembrane domain of the protein. However, due to structural similarities, the nomenclature of antigenic sites used for the fusion protein of respiratory syncytial virus (RSV), another member of the Pneumoviridae family, can be used to more accurately describe protein regions. DIIIa experiences the greatest conformational rearrangement when the fusion peptide is released from the trimeric inner cavity, and the HRA forms a three-coil bundle simultaneously as the fusion peptide inserts into the target membrane. Finally, HRB associates around the outside of the HRA bundle to form 6HB in the postfusion conformation.

[0085] Recently, stabilization of the prefusion conformation of class I fusion proteins has yielded promising results as vaccine antigens in clinical trials, and this approach has been used for both RSV vaccines and SARS-CoV-2 vaccines (Baden et al., 2020, Keech et al., 2020, Williams et al., 2020, Crank et al., 2019). RSV shares approximately 33% sequence identity with hMPV, and these two prefusion structures are highly similar (Battles et al., 2017, van den Hoogen et al., 2002). Based on the resolved prefusion structures, a few different strategies have been used to stabilize RSV F in its prefusion conformation, including the introduction of prolines, disulfide bonds, and cavity-filling substitutions (Joyce et al., 2016, Krarup et al., 2015, McLellan et al., 2013). In the case of DS-Cav1, the internal cavity was more optimally filled by hydrophobic residue substitutions, and disulfide bonds were introduced in the fusion peptide region. In the case of PR-DM, proline residues were introduced to prevent the loop regions from restructuring into extended alpha helices seen in the postfusion conformation. In RSV F, regions of charge repulsion were also identified and reduced. Similarly, the resolved prefusion structure enabled stabilization of coronaviruses by introducing two proline substitutions (S-2P) into the hinge region. There are also examples within the fields of HIV (SOSIP) and influenza where the introduction of stabilizing mutations has led to well-functioning prefusion reagents.

[0086] Recently, using the knowledge obtained from RSV F research, hMPV F was stabilized in its pre-fusion conformation. First, the F2 / F1 cleavage site sequence "RQSR" was replaced with the polybasic "RRRR" sequence to enable efficient cleavage by furin-like proteases in the production cells. Next, mimicking the RSV F stabilization strategy (Krarup et al., 2015), prolines were introduced into the helix-loop-helix region in F1 at the membrane-distal trimer apex. This engineering strategy allowed the obtaining of the pre-fusion crystal structure of hMPV F, but suggested that protein expression was insufficient and further engineering was required (Battles et al., 2017). Furthermore, previous serum depletion assays and mouse immunization experiments showed no significant antigenic difference between pre-fusion and post-fusion hMPV F (Battles et al., 2017). In contrast, pre-fusion RSV F induced a stronger neutralizing antibody response than post-fusion RSV F, and serum depletion experiments demonstrated that most of the RSV neutralizing activity in human sera binds exclusively to the pre-fusion conformation (Sastre et al., 2005, Magro et al., 2012). These data suggested that more stable pre-fusion hMPV F constructs were needed to investigate these discrepant results.

[0087] For this purpose, an F protein stabilization strategy by mutating the coding sequence of the F protein is demonstrated herein. Using the published pre-fusion hMPV F structure, the introduction of additional amino acid substitutions was engineered. Combinations of multiple beneficial substitutions were found to have an additive effect on the desired protein properties. The details of the mutations analyzed and provided herein are shown in Table 1 below. The mutant proteins were expressed as detailed in the Examples, and the amount of the produced protein-trimer complex was determined.

[0088] (Table 1) F Protein Substitutions and Mutations TIFF2025102893000001.tif225158TIFF2025102893000002.tif206158TIFF2025102893000003.tif211163TIFF2025102893000004.tif100163

[0089] IV. Pharmaceutical Preparation The present disclosure provides a pharmaceutical composition comprising an engineered hMPV F protein. Such a composition can be used to stimulate an immune response, such as part of a vaccine formulation.

[0090] When a nucleic acid molecule encoding an engineered hMPV F protein is used in a pharmaceutical composition, the nucleic acid molecule may comprise or consist of covalently linked together deoxyribonucleotides and / or ribonucleotides, or analogs thereof. The nucleic acid molecules described herein generally contain phosphodiester bonds, but in some cases may contain at least one different bond, such as phosphoramidate, phosphorothioate, phosphorodithioate, or O-methylphosphoramidite bonds, as well as nucleic acid analogs that may have peptide nucleic acid backbones and linkages. Mixtures of native polynucleotides and analogs can be made, but alternatively, mixtures of different polynucleotide analogs, as well as mixtures of native polynucleotides and analogs, may be made. The nucleic acid molecule may contain modified nucleotides such as methylated nucleotides and nucleotide analogs. Modifications to the nucleotide structure, if present, may be imparted before or after polymerization of the polymer. Non-nucleotide components may be interspersed within the nucleotide sequence. The polynucleotide may be further modified after polymerization, for example, by conjugation with a labeling component. The term also includes both double-stranded and single-stranded molecules. Unless otherwise specified or required, the term polynucleotide includes both the double-stranded form and each of the two complementary single-stranded forms known or predicted to constitute the double-stranded form. Nucleic acid molecules are composed of a specific sequence of the four nucleotide bases, adenine (A), cytosine (C), guanine (G), thymine (T), and uracil (U) in place of thymine when the polynucleotide is RNA. Thus, the term "nucleic acid sequence" is the alphabetic representation of a nucleic acid molecule. Unless otherwise indicated, a particular nucleic acid sequence also implicitly encompasses not only the explicitly shown sequence, but also its conservatively modified variants (e.g., degenerate codon substitutions) and complementary sequences. Specifically, degenerate codon substitutions may be achieved by generating a sequence in which the third position of one or more selected (or all) codons is substituted with a mixed base and / or deoxyinosine residue.

[0091] In some embodiments, the nucleic acids of the present disclosure comprise one or more modified nucleosides comprising a modified sugar moiety. Such compounds comprising one or more sugar-modified nucleosides can have desirable properties such as enhanced nuclease stability or increased binding affinity to a target nucleic acid compared to oligonucleotides comprising only nucleosides with native sugar moieties. In some embodiments, the modified sugar moiety is a substituted sugar moiety. In some embodiments, the modified sugar moiety is a sugar surrogate. Such sugar surrogates may include one or more substitutions corresponding to the substitution of the substituted sugar moiety.

[0092] In some embodiments, the modified sugar moiety is a substituted sugar moiety comprising one or more non-bridging sugar substituents including, but not limited to, substituents at the 2'-position and / or 5'-position. Examples of suitable sugar substituents at the 2'-position include, but are not limited to, 2'-F, 2'-OCH3 ("OMe" or "O-methyl"), and 2'-O(CH2)2OCH3 ("MOE"). In certain embodiments, the sugar substituent at the 2'-position is allyl, amino, azido, thio, O-allyl, O--C1~C 10 alkyl, O--C1~C 10 substituted alkyl, OCF3, O(CH2)2SCH3, O(CH2)2--O--N(Rm)(Rn), and O--CH2--C(=O)--N(Rm)(Rn), wherein each Rm and Rn is independently H or substituted or unsubstituted C1~C 10 alkyl. Examples of sugar substituents at the 5'-position include, but are not limited to, 5'-methyl (R or S), 5'-vinyl, and 5'-methoxy. In some embodiments, the substituted sugar comprises more than one non-bridging sugar substituent, e.g., a T-F-5'-methyl sugar moiety (see, e.g., PCT International Application WO2008 / 101157 for additional 5',2'-bis-substituted sugar moieties and nucleosides).

[0093] Nucleosides containing a 2'-substituted sugar moiety are referred to as 2'-substituted nucleosides. In some embodiments, the 2'-substituted nucleoside comprises a 2'-substituent selected from halo, allyl, amino, azide, SH, CN, OCN, CF3, OCF3, O, S, or N(Rm)-alkyl; O, S, or N(Rm)-alkenyl; O, S or N(Rm)-alkynyl; O-alkylenyl-O-alkyl, alkynyl, alkaryl, aralkyl, O-alkyl, O-aralkyl, O(CH2)2SCH3, O(CH2)2--O--N(Rm)(Rn), or O--CH2--C(=O)--N(Rm)(Rn), wherein each Rm and Rn is independently H, an amino protecting group, or substituted or unsubstituted C1-C 10 alkyl. These 2'-substituents may be further substituted with one or more substituents independently selected from hydroxyl, amino, alkoxy, carboxy, benzyl, phenyl, nitro (NO2), thiol, thioalkoxy (S-alkyl), halogen, alkyl, aryl, alkenyl, and alkynyl.

[0094] In some embodiments, the 2'-substituted nucleoside comprises a 2'-substituent selected from F, NH2, N3, OCF3, O--CH3, O(CH2)3NH2, CH2-CH=CH2, O--CH2-CH=CH2, OCH2CH2OCH3, O(CH2)2SCH3, O--(CH2)2--O--N(Rm)(Rn), O(CH2)2O(CH2)2N(CH3)2, and N-substituted acetamide (O--CH2--C(=O)--N(Rm)(Rn), wherein each Rm and Rn is independently H, an amino protecting group, or substituted or unsubstituted C1-C 10 alkyl.

[0095] In some embodiments, the 2'-substituted nucleoside comprises a sugar moiety comprising a 2'-substituent selected from F, OCF3, O--CH3, OCH2CH2OCH3, O(CH2)2SCH3, O(CH2)2--O--N(CH3)2, --O(CH2)2O(CH2)2N(CH3)2, and O--CH2--C(=O)--N(H)CH3.

[0096] In some embodiments, the 2'-substituted nucleoside comprises a sugar moiety comprising a 2'-substituent selected from F, O--CH3, and OCH2CH2OCH3.

[0097] In some embodiments, the nucleosides of the disclosure comprise one or more unmodified nucleobases. In certain embodiments, the nucleosides of the disclosure comprise one or more modified nucleobases.

[0098] In some embodiments, the modified nucleobases are selected from universal bases, hydrophobic bases, promiscuous bases, size-expanded bases, and fluorinated bases as defined herein. 5-Substituted pyrimidines, 6-azapyrimidines, and N-2, N-6, and O-6 substituted purines include, as defined herein, 2-aminopropyladenine, 5-propynyluracil; 5-propynylcytosine; 5-hydroxymethylcytosine, xanthine, hypoxanthine, 2-aminoadenine, 6-methyl and other alkyl derivatives of adenine and guanine, 2-propyl and other alkyl derivatives of adenine and guanine, 2-thiouracil, 2-thiothymine, and 2-thiocytosine, 5-halouracil and cytosine, 5-propynyl(CH3)uracil and cytosine and other alkynyl derivatives of pyrimidine bases, 6-azouracil, cytosine, and thymine, 5-uracil(pseudouracil), 4-thiouracil, 8-halo, 8-amino, 8-thiol, 8-thioalkyl, 8-hydroxyl and other 8-substituted adenines and guanines, 5-halo, particularly 5-bromo, 5-trifluoromethyl and other 5-substituted uracils and cytosines, 7-methylguanine and 7-methyladenine, 2-F-adenine, 2-amino-adenine, 8-azaguanine and 8-azaadenine, 7-deazaguanine and 7-deazaadenine, 3-deazaguanine and 3-deazaadenine, universal bases, hydrophobic bases, promiscuous bases, size-expanded bases, and fluorinated bases. Further modified nucleobases include tricyclic pyrimidines such as phenoxazine cytidine ([5,4-b][1,4]benzoxazin-2(3H)-one), phenothiazine cytidine (1H-pyrimido[5,4-b][1,4]benzothiazin-2(3H)-one), G-clamps such as substituted phenoxazine cytidine (e.g., 9-(2-aminoethoxy)-H-pyrimido[5,4-13][1,4]benzoxazin-2(3H)-one), carbazole cytidine (2H-pyrimido[4,5-b]indol-2-one), pyridoindole cytidine (H-pyrido[3’,2’:4,5]pyrrolo[2,3-d]pyrimidin-2-one).Modified nucleobases also include those in which the purine or pyrimidine base is replaced with another heterocycle, such as 7-deaza-adenine, 7-deazaguanosine, 2-aminopyridine, and 2-pyridone. Further nucleobases include those disclosed in U.S. Patent 3,687,808 and those disclosed in The Concise Encyclopedia Of Polymer Science And Engineering, Kroschwitz, J.I., Ed., John Wiley & Sons, 1990, 858-859.

[0099] Representative U.S. patents that teach the preparation of certain of the above-described modified nucleobases, as well as other modified nucleobases, include, but are not limited to, U.S. Patents 3,687,808, 4,845,205, 5,130,302, 5,134,066, 5,175,273, 5,367,066, 5,432,272, 5,457,187, 5,459,255, 5,484,908, 5,502,177, 5,525,711, 5,552,540, 5,587,469, 5,594,121, 5,596,091, 5,614,617, 5,645,985, 5,681,941, 5,750,692, 5,763,588, 5,830,653, and 6,005,096, each of which is incorporated herein by reference in its entirety.

[0100] In addition, additional modifications may be made at other positions on the oligonucleotide, particularly at the 3'-position of the sugar on the 3'-terminal nucleotide and the 5'-position of the 5'-terminal nucleotide. For example, one additional modification of the ligand-conjugated oligonucleotides of the present disclosure involves chemically linking one or more additional non-ligand moieties or conjugates that enhance the activity, cellular distribution, or cellular uptake of the oligonucleotide to the oligonucleotide. Such moieties include, but are not limited to, lipid moieties such as cholesterol moieties, cholic acid, thioethers such as hexyl-5-tritylthiol, thiocolesterol, aliphatic chains such as dodecanediol or undecyl residues, phospholipids such as di-hexadecyl-rac-glycerol or triethylammonium 1,2-di-O-hexadecyl-rac-glycero-3-H-phosphonate, polyamines or polyethylene glycol chains, or adamantane acetic acid, palmitoyl moieties, or octadecylamine or hexylamino-carbonyl-oxy cholesterol moieties. In some embodiments, the nucleic acid molecule encoding the chimeric hMPV / RSV F protein is a modified RNA, such as a modified mRNA. Modified (m)RNA contemplates certain chemical modifications that confer increased stability and low immunogenicity to the mRNA, thereby facilitating the expression of therapeutically important proteins. For example, N1-methyl-pseudouridine (N1mΨ) is superior to several other nucleoside modifications and combinations thereof in terms of translational ability. In some embodiments, the (m)RNA molecule used herein may have uracil replaced with pseudouridine, such as 1-methyl-3'-pseudouridinyl base. In some embodiments, some of the uracils are replaced, while in other embodiments, all of the uracils are replaced. The (m)RNA may include a 5' cap, a 5' UTR element, an optionally codon-optimized open reading frame, a 3' UTR element, as well as a poly(A) sequence and / or a polyadenylation signal.

[0101] The nucleic acid molecule, whether native or modified, may be delivered as a naked nucleic acid molecule or in a delivery vehicle such as a lipid nanoparticle. The lipid nanoparticle may contain one or more nucleic acids present at a weight ratio of about 5:1 to about 1:100 relative to the lipid nanoparticle. In some embodiments, the weight ratio of nucleic acid to lipid nanoparticle is about 5:1, 2.5:1, 1:1, 1:5, 1:10, 1:15, 1:20, 1:25, 1:30, 1:35, 1:40, 1:45, 1:50, 1:60, 1:70, 1:80, 1:90, or 1:100, or any value derivable therein.

[0102] In some embodiments, the lipid nanoparticles used herein may contain 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 lipids. These lipids may include groups having one or more hydrophobic groups such as triglycerides, phospholipids, steroids or sterols, PEGylated lipids, or ionizable groups such as alkylamines and alkyl groups having 6 or more carbon atoms.

[0103] In some aspects of the present disclosure, the lipid nanoparticles are mixed with one or more steroids or steroid derivatives. In some embodiments, the steroid or steroid derivative includes any steroid or steroid derivative. As used herein, in some embodiments, the term "steroid" is a class of compounds having a 4-ring, 17-carbon cyclic structure, which may further include one or more substitutions including alkyl groups, alkoxy groups, hydroxy groups, oxo groups, acyl groups, or double bonds between two or more carbon atoms.

[0104] In some aspects of the present disclosure, the lipid nanoparticles are mixed with one or more PEGylated lipids (or PEG lipids). In some embodiments, the present disclosure includes using any lipid to which a PEG group is attached. In some embodiments, the PEG lipid is a diglyceride that also includes a PEG chain attached to a glycerol group. In other embodiments, the PEG lipid is a compound containing one or more C6-C24 long-chain alkyl or alkenyl groups or C6-C24 fatty acid groups attached to a linker group having a PEG chain. Some non-limiting examples of PEG lipids include PEG-modified phosphatidylethanolamine and phosphatidic acid, PEG-ceramide conjugates, PEG-modified dialkylamines and PEG-modified 1,2-diacetyloxypropane-3-amines, PEG-modified diacylglycerols and dialkylglycerols. In some embodiments, PEG-modified distearoyl phosphatidylethanolamine or PEG-modified dimyristoyl-sn-glycerol. In some embodiments, the PEG modification is measured by the molecular weight of the PEG component of the lipid. In some embodiments, the PEG modification has a molecular weight of from about 100 to about 15,000. In some embodiments, the molecular weight is from about 200 to about 500, from about 400 to about 5,000, from about 500 to about 3,000, or from about 1,200 to about 3,000. The molecular weight of the PEG modification is from about 100, 200, 400, 500, 600, 800, 1,000, 1,250, 1,500, 1,750, 2,000, 2,250, 2,500, 2,750, 3,000, 3,500, 4,000, 4,500, 5,000, 6,000, 7,000, 8,000, 9,000, 10,000, 12,500 to about 15,000. Some non-limiting examples of lipids that can be used in the present disclosure are taught by U.S. Patent 5,820,873, WO2010 / 141069, or U.S. Patent 8,450,298, which are hereby incorporated by reference in their entirety.

[0105] In some aspects of the present disclosure, the lipid nanoparticles are mixed with one or more phospholipids. In some embodiments, any lipid that also contains a phosphate group. In some embodiments, the phospholipid has one or two long-chain C6-C 24 It is a structure containing an alkyl or alkenyl group, glycerol or sphingosine, one or two phosphate groups, and optionally an organic small molecule. In some embodiments, the organic small molecule is an amino acid, a sugar, or an amino-substituted alkoxy group such as choline or ethanolamine. In some embodiments, the phospholipid is phosphatidylcholine. In some embodiments, the phospholipid is distearoyl phosphatidylcholine or dioleoyl phosphatidylethanolamine. In some embodiments, other zwitterionic lipids are used, where zwitterionic lipids are defined as lipids and lipid-like molecules having both a positive charge and a negative charge.

[0106] In some aspects of the present disclosure, lipid nanoparticles are provided that contain a compound containing a lipophilic component and a cationic component, where the cationic component is ionizable. In some embodiments, the ionizable cationic lipid is protonated at physiological pH, but can be deprotonated at a pH above 8, 9, 10, 11, or 12 and contains one or more groups that have no charge. The ionizable cationic group may contain one or more protonatable amines that can form a cationic group at physiological pH. The ionizable cationic lipid compound may also contain one or more lipid components such as two or more fatty acids having a C6-C 24 alkyl or alkenyl carbon group. These lipid groups may be bonded via an ester bond or may be further attached to a sulfur atom via a Michael addition reaction. In some embodiments, these compounds can be dendrimers, dendrons, polymers, or combinations thereof.

[0107] In some aspects of the present disclosure, there is provided a composition comprising a compound containing a lipophilic component and a cationic component, wherein the cationic component is ionizable. In some embodiments, an ionizable cationic lipid refers to a lipid and lipid-like molecule having a nitrogen atom capable of acquiring a charge (pKa). These lipids may be known in the literature as cationic lipids. These molecules having an amino group typically have 2 to 6 hydrophobic chains, often C6 to C 24 alkyl or alkenyl, such as alkyl or alkenyl groups, but may have at least 1 or more than 6 tails.

[0108] In some embodiments, the amount of lipid nanoparticles having a nucleic acid molecule encapsulated therein in the pharmaceutical composition is from about 0.1 w / w% to about 50 w / w%, from about 0.25 w / w% to about 25 w / w%, from about 0.5 w / w% to about 20 w / w%, from about 1 w / w% to about 15 w / w%, from about 2 w / w% to about 10 w / w%, from about 2 w / w% to about 5 w / w%, or from about 6 w / w% to about 10 w / w%. In some embodiments, the amount of lipid nanoparticles having a nucleic acid molecule encapsulated therein in the pharmaceutical composition is about 0.1 w / w%, 0.25 w / w%, 0.5 w / w%, 1 w / w%, 2.5 w / w%, 5 w / w%, 7.5 w / w%, 10 w / w%, 15 w / w%, 20 w / w%, 25 w / w%, 30 w / w%, 35 w / w%, 40 w / w%, 45 w / w%, 50 w / w%, 55 w / w%, 60 w / w%, 65 w / w%, 70 w / w%, 75 w / w%, 80 w / w%, 85 w / w%, 90 w / w% to about 95 w / w%, or any range derivable therein.

[0109] In some embodiments, the present disclosure includes one or more sugars incorporated in a pharmaceutical composition. In some embodiments, the sugars used herein are saccharides. These saccharides may be used to function as cryoprotectants that protect the pharmaceutical composition from destabilization during the drying process. These water-soluble excipients include carbohydrates or saccharides, such as disaccharides like sucrose, trehalose, or lactose, trisaccharides like raffinose - composed of fructose, glucose, galactose, etc., polysaccharides like starch or cellulose, or sugar alcohols like xylitol, sorbitol, or mannitol. In some embodiments, these excipients are solids at room temperature. Some non - limiting examples of sugar alcohols include erythritol, threitol, arabinitol, xylitol, ribitol, mannitol, sorbitol, galactitol, fucitol, iditol, inositol, volemitol, isomalt, maltitol, lactitol, maltotritol, maltotetraitol, or polyglycitols.

[0110] In some embodiments, the amount of sugar in the pharmaceutical composition is from about 25 w / w% to about 98 w / w%, from 40 w / w% to about 95 w / w%, from 50 w / w% to about 90 w / w%, from 50 w / w% to about 70 w / w%, or from about 80 w / w% to about 90 w / w%. In some embodiments, the amount of sugar in the pharmaceutical composition is about 10 w / w%, 15 w / w%, 20 w / w%, 25 w / w%, 30 w / w%, 35 w / w%, 40 w / w%, 45 w / w%, 50 w / w%, 52.5 w / w%, 55 w / w%, 57.5 w / w%, 60 w / w%, 62.5 w / w%, 65 w / w%, 67.5 w / w%, 70 w / w%, 75 w / w%, 80 w / w%, 82.5 w / w%, 85 w / w%, 87.5 w / w%, 90 w / w% to about 95 w / w%, or any range derivable therein.

[0111] In some embodiments, the pharmaceutically acceptable polymer is a copolymer. The pharmaceutically acceptable polymer may further comprise 1, 2, 3, 4, 5, or 6 subunits of different individual types of polymer subunits. These polymer subunits may include polyoxypropylene, polyoxyethylene, or similar subunits. In particular, the pharmaceutically acceptable polymer may comprise at least one hydrophobic subunit and at least one hydrophilic subunit. In particular, the copolymer may have hydrophilic subunits on each side of the hydrophobic unit. The copolymer may have a hydrophilic subunit that is polyoxyethylene and a hydrophobic subunit that is polyoxypropylene.

[0112] In some embodiments, an expression cassette is used to express the engineered hMPV F protein for subsequent purification and delivery to cells / subjects or for direct use in a virus-based delivery approach. Provided herein is an expression vector containing one or more nucleic acids encoding the engineered hMPV F protein.

[0113] Expression requires that appropriate signals be provided within the vector and that it contain various regulatory elements such as enhancers / promoters from both viral and mammalian sources that drive the expression of the engineered hMPV F protein intracellularly. Throughout this application, the term "expression cassette" is intended to include any kind of gene construct that contains a nucleic acid encoding a gene product, where a part or all of the coding sequence of the nucleic acid can be transcribed and translated, i.e., is under the control of a promoter. A "promoter" refers to a DNA sequence that is recognized by the synthetic machinery of the cell or introduced synthetic machinery and is required to initiate the specific transcription of a gene. The phrase "under transcriptional control" means that the promoter is in the correct position and orientation with respect to the nucleic acid to control the initiation of RNA polymerase and the expression of the gene. An "expression vector" is intended to include an expression cassette contained within a gene construct that is capable of replication and thus contains one or more of an origin of replication, a transcription termination signal, a poly-A region, a selectable marker, and a multiple cloning site.

[0114] The term promoter will be used herein to refer to a group of transcriptional control modules clustered around the start site for RNA polymerase II. Most of the thinking about how promoters are organized has come from the analysis of several viral promoters, including the analysis of the HSV thymidine kinase (tk) and the SV40 early transcription unit. These studies, enhanced by more recent efforts, have shown that promoters are composed of individual functional modules, each consisting of approximately 7 - 20 bp of DNA and containing one or more recognition sites for transcriptional activator or repressor proteins.

[0115] At least one module in each promoter functions to position the start site for RNA synthesis. The best-known example of this is the TATA box, but in some promoters lacking a TATA box, such as the promoter for the mammalian terminal deoxynucleotidyl transferase gene and the promoter for the SV40 late gene, the individual element itself overlapping the start site helps to fix the starting point.

[0116] Additional promoter elements regulate the frequency of transcription initiation. Typically, these are located in the region 30 - 110 bp upstream of the start site, but in recent years, some promoters have been shown to contain functional elements also downstream of the start site. The spacing between promoter elements is often flexible, so that promoter function is maintained even when the elements are inverted or moved relative to each other. In the tk promoter, the spacing between promoter elements can be increased up to a spacing of 50 bp, after which activity begins to decline. Depending on the promoter, individual elements can function either cooperatively or independently to activate transcription.

[0117] In certain embodiments, viral promoters such as the human cytomegalovirus (CMV) immediate early gene promoter, the SV40 early promoter, the Rous sarcoma virus long terminal repeat, the rat insulin promoter, and glyceraldehyde-3-phosphate dehydrogenase are used to obtain high levels of expression of the coding sequence of interest. The use of other viral or mammalian cell or bacteriophage promoters well known in the art for achieving expression of the coding sequence of interest is also contemplated, provided that the expression level is sufficient for a given purpose. By using promoters with known characteristics, the expression level and pattern of the protein of interest after transfection or transformation can be optimized. Furthermore, the selection of a promoter that is regulated in response to specific physiological signals can allow for inducible expression of the gene product.

[0118] Enhancers are genetic elements that increase transcription from a promoter and are located at distant positions on the same DNA molecule. Enhancers are organized in a manner quite similar to promoters. That is, they are composed of many individual elements, each of which binds one or more transcription proteins. The fundamental difference between enhancers and promoters is functional. As a whole, the enhancer region must be able to stimulate transcription from a distance, which need not apply to the promoter region or its components. On the other hand, a promoter must have one or more elements that direct the initiation of RNA synthesis at a specific site and in a specific orientation, which enhancers lack. Promoters and enhancers often overlap and are contiguous and often appear to have very similar modular compositions.

[0119] The following is a list of promoters / enhancers and inducible promoters / enhancers that can be used in combination with a nucleic acid encoding a gene of interest in an expression construct. Additionally, any combination of promoters / enhancers (from the eukaryotic promoter database EPDB) can also be used to drive gene expression. Eukaryotic cells can support cytoplasmic transcription from certain bacterial promoters when an appropriate bacterial polymerase is provided either as part of the delivery complex or as an additional gene expression construct.

[0120] Promoters and / or enhancers can be, for example, immunoglobulin light chain, immunoglobulin heavy chain, T cell receptor, HLA DQ a and / or DQ β, β-interferon, interleukin-2, interleukin-2 receptor, MHC class II 5, MHC class II HLA-Dra, β-actin, muscle creatine kinase (MCK), prealbumin (transthyretin), elastase I, metallothionein (MTII), collagenase, albumin, α-fetoprotein, t-globin, β-globin, c-fos, c-HA-ras, insulin, neural cell adhesion molecule (NCAM), α1-antitrypsin, H2B (TH2B) histone, mouse and / or type I collagen, glucose-regulated protein (GRP94 and GRP78), rat growth hormone, human serum amyloid A (SAA), troponin I (TN I), platelet-derived growth factor (PDGF), SV40, polyoma, retrovirus, papillomavirus, hepatitis B virus, human immunodeficiency virus, cytomegalovirus (CMV), and simian leukemia virus.

[0121] When cDNA inserts are used, typically it will be desirable to include a polyadenylation signal to achieve proper polyadenylation of the gene transcript. Any polyadenylation sequence such as the human growth hormone and SV40 polyadenylation signals may be used. Also, terminators are contemplated as elements of the expression cassette. These elements can serve to enhance the message level and minimize read-through to other sequences from the cassette.

[0122] There are several methods by which an expression vector can be introduced into cells. In certain embodiments, the expression construct includes a virus or an engineered construct derived from a viral genome. Certain viruses are attractive candidates for the transfer of foreign genes into mammalian cells due to their ability to enter cells via receptor-mediated endocytosis and integrate into the host cell genome to stably and efficiently express viral genes. These have a relatively low capacity for foreign DNA sequences and have a restricted host spectrum. Furthermore, their oncogenic potential and cytopathic effects in permissive cells raise concerns about safety. They can accommodate a maximum of 8 kB of foreign genetic material but can be easily introduced into various cell lines and experimental animals.

[0123] One method for in vivo delivery is the use of an adenovirus expression vector. By "adenovirus expression vector" is meant a construct that contains (a) adenovirus sequences sufficient to support packaging of the construct and (b) to express the engineered hMPV F protein cloned therein. In this context, expression does not require that the gene product be synthesized.

[0124] The expression vector includes an adenovirus in genetically engineered form. Knowledge of the genetic makeup of adenovirus, a 36 kB linear double-stranded DNA virus, allows for the replacement of large fragments of adenovirus DNA with foreign sequences up to 7 kB. In contrast to retroviruses, adenovirus infection of host cells does not result in chromosomal integration because adenovirus DNA can replicate in an episomal fashion without potential genotoxicity. Also, adenoviruses are structurally stable and no genome rearrangement has been detected after large-scale amplification. Adenoviruses can infect virtually all epithelial cells, regardless of the stage of their cell cycle. To date, adenovirus infections are thought to be associated only with mild diseases such as acute respiratory diseases in humans.

[0125] Adenoviruses are particularly suitable for use as gene transfer vectors because of their medium genome size, ease of manipulation, high titer, broad target cell range, and high infectivity. Both ends of the viral genome contain inverted terminal repeats (ITRs) of 100-200 base pairs, which are cis elements necessary for viral DNA replication and packaging. The early (E) and late (L) regions of the genome contain different transcription units that are separated by the initiation of viral DNA replication. The E1 region (E1A and E1B) encodes proteins that are responsible for the regulation of transcription of the viral genome and a few cellular genes. Expression of the E2 region (E2A and E2B) results in the synthesis of these proteins for viral DNA replication. These proteins are involved in DNA replication, late gene expression, and host cell shutoff. The products of the late genes, including most of the viral capsid proteins, are expressed only after significant processing of a single primary transcript produced by the major late promoter (MLP). The MLP (located at 16.8 m.u.) is particularly efficient during the late stages of infection, and all mRNAs produced from this promoter carry the 5'-tripartite leader (TPL) sequence, making them preferred mRNAs for translation. In one system, recombinant adenoviruses are generated from homologous recombination between a shuttle vector and a proviral vector. Due to the possibility of recombination between two proviral vectors, wild-type adenoviruses can be generated from this process. Therefore, it is essential to isolate a single clone of the virus from an individual plaque and examine its genomic structure.

[0126] The generation and propagation of current adenovirus vectors with replication defects rely on a specific helper cell line called 293, which was transformed from human fetal kidney cells by Ad5 DNA fragments and constitutively expresses the E1 protein. Since the E3 region is not essential for the adenovirus genome, current adenovirus vectors carry foreign DNA in either the E1, D3, or both regions with the help of 293 cells. Originally, adenovirus can package approximately 105% of the wild-type genome and provide an extra capacity for about 2 kb of DNA. When combined with approximately 5.5 kb of DNA that can be replaced in the E1 and E3 regions, the maximum capacity of current adenovirus vectors is less than 7.5 kb, or about 15% of the total length of the vector. More than 80% of the adenovirus genome remains in the vector backbone, becoming a source of vector-derived cytotoxicity. Also, the replication defect of E1-deleted viruses is incomplete.

[0127] The helper cell line can be derived from human cells such as human fetal kidney cells, muscle cells, hematopoietic cells, or other mesenchymal or epithelial human fetal cells. Alternatively, the helper cells can be derived from cells of other mammalian species that are permissive for human adenovirus. Such cells include, for example, Vero cells or other mesenchymal or epithelial rhesus fetal cells. As described above, the preferred helper cell line is 293.

[0128] The adenoviruses of the present disclosure are replication-deficient or at least conditionally replication-deficient. The adenoviruses can be of any of 42 different known serotypes or subgroups A - F. Adenovirus type 5 of subgroup C is one exemplary starting material that can be used to obtain a conditionally replication-deficient adenovirus vector for use in the present invention.

[0129] Other viral vectors may also be used as the expression constructs in the present disclosure. Vectors derived from viruses such as vaccinia virus, adeno-associated virus (AAV), and herpes virus may also be used. They present several attractive features for various mammalian cells.

[0130] In an embodiment, in a particular embodiment, the vector is an AAV vector. AAV is a small virus that infects humans and several other primate species. AAV is not currently known to cause disease. The lack of apparent pathogenicity of this virus is further supported by the fact that it elicits a very mild immune response. In many cases, AAV vectors integrate into the host cell genome, which can be important for certain applications but can also have unwanted consequences. Gene therapy vectors using AAV can infect both dividing and quiescent cells and persist episomally without integrating into the host cell genome, whereas in the native virus, some integration of the gene carried by the virus into the host genome occurs. These features make AAV a very attractive candidate for creating viral vectors for gene therapy and for creating isogenic human disease models. Recent human clinical trials using AAV for gene therapy in the retina have shown promise. AAV belongs to the genus Dependoparvovirus, which in turn belongs to the family Parvoviridae. This virus is a small (20 nm) replication-deficient virus without an envelope.

[0131] Wild-type AAV has attracted considerable interest among gene therapy researchers for several characteristics. Among these, the most prominent is the apparent lack of pathogenicity of this virus. It can also infect non-dividing cells and has the ability to stably integrate into the host cell genome at a specific site on human chromosome 19 (designated AAVS1). Due to this characteristic, it is somewhat more predictable than retroviruses, which pose a threat of random insertion and mutagenesis that can sometimes lead to the development of cancer. The AAV genome is most frequently integrated into the site mentioned, while random integration into the genome occurs at a very low frequency. However, the development of AAV as a gene therapy vector has eliminated this integration ability by removing rep and cap from the vector DNA. The desired gene, along with a promoter to drive gene transcription, is inserted between the inverted terminal repeats (ITRs) that assist in concatemer formation in the nucleus after the single-stranded vector DNA is converted to double-stranded DNA by the host cell DNA polymerase complex. AAV-based gene therapy vectors form episomal concatemers in the host cell nucleus. In non-dividing cells, these concatemers remain intact throughout the life of the host cell. In dividing cells, AAV DNA is lost during cell division because episomal DNA is not replicated with the host cell DNA. Random integration of AAV DNA into the host genome is detectable but occurs at a very low frequency. AAV also exhibits very low immunogenicity, which appears to be limited to the production of neutralizing antibodies, while they do not induce a clearly defined cytotoxic response. This characteristic, along with the ability to infect quiescent cells, demonstrates their superiority over adenoviruses as vectors for human gene therapy.

[0132] The AAV genome consists of single-stranded deoxyribonucleic acid (ssDNA), either plus-strand or minus-strand, which is approximately 4.7 kilobases in length. This genome contains inverted terminal repeat (ITR) sequences at both ends of the DNA strand, as well as two open reading frames (ORFs), rep and cap. The former is composed of four overlapping genes that encode the Rep proteins required throughout the AAV life cycle, and the latter contains the overlapping nucleotide sequences of the capsid proteins VP1, VP2, and VP3, which interact together to form an icosahedral symmetric capsid.

[0133] The inverted terminal repeat (ITR) sequences each contain 145 bases. They are so named because of their symmetry, which has been shown to be required for efficient multiplication of the AAV genome. The feature of these sequences that confers this property is their ability to form hairpins, which contributes to so-called self-priming, enabling primer-independent synthesis of the second DNA strand. ITRs have also been shown to be required for both integration of AAV DNA into the host cell genome (chromosome 19 in humans) and its rescue therefrom, as well as for efficient capsid formation of AAV DNA in combination with the generation of fully assembled deoxyribonuclease-resistant AAV particles.

[0134] Regarding gene therapy, the ITRs appear to be the only sequences required in cis adjacent to the therapeutic gene, and the structural (cap) and packaging (rep) proteins can be delivered in trans. Based on this assumption, many methods have been established for the efficient production of recombinant AAV (rAAV) vectors containing reporter or therapeutic genes. However, it has also been reported that the ITRs are not the only elements required in cis for efficient replication and capsid formation. A few research groups have identified a sequence called the cis-acting Rep-dependent element (CARE) that lies within the coding sequence of the rep gene. CARE has been shown to enhance replication and capsid formation when present in cis.

[0135] In some aspects, the present disclosure provides a pharmaceutical composition containing one or more salts. The salts can be inorganic potassium or sodium salts such as potassium chloride, sodium chloride, dibasic potassium phosphate, monobasic potassium phosphate, dibasic sodium phosphate, or monobasic sodium phosphate. The pharmaceutical composition may contain one or more phosphates, such as for generating a phosphate buffer. The phosphate buffer may contain each of the phosphates to buffer the solution to a pH of about 6.8, 6.9, 7.0, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9, or 8.0, or any range derivable therein.

[0136] In some embodiments, the present disclosure includes one or more excipients formulated in a pharmaceutical composition. An "excipient" refers to a pharmaceutically acceptable carrier, which is a relatively inert substance used to facilitate the administration or delivery of an API to a subject or to facilitate the processing of an API into a pharmaceutical formulation that can be pharmaceutically used for delivery to a site of action within the subject. Further, these compounds may be used as diluents to obtain a dosage that can be easily measured or administered to a patient. Non-limiting examples of excipients include polymers, stabilizers, surfactants, surface modifiers, solubilizers, buffers, encapsulating agents, antioxidants, preservatives, nonionic wetting or clarifying agents, thickeners, and absorption enhancers.

[0137] In a specific embodiment, the term "pharmaceutically acceptable" means approved by a regulatory agency of the federal or state government or listed in the United States Pharmacopeia or other generally recognized pharmacopeias for use in animals, and more particularly in humans. The term "carrier" refers to a diluent, excipient, or vehicle administered with a therapeutic agent. Such a pharmaceutical carrier can be a sterile liquid such as water and may preferably contain adjuvants. When the pharmaceutical composition is administered by injection such as intramuscular injection, water is a specific carrier. Aqueous solutions of saline as well as dextrose and glycerol may also be used particularly as liquid carriers for injectable solutions. Other suitable pharmaceutical excipients include starch, glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silica gel, sodium stearate, glycerol monostearate, talc, sodium chloride, dried skim milk, glycerol, propylene, glycol, water, ethanol, and the like.

[0138] The composition can also contain, if desired, small amounts of wetting or emulsifying agents, or pH buffers. These compositions can take the form of solutions, suspensions, emulsions, tablets, pills, capsules, powders, sustained-release formulations, etc. Oral formulations can contain standard carriers such as pharmaceutical-grade mannitol, lactose, starch, magnesium stearate, sodium saccharin, cellulose, magnesium carbonate, and the like. Examples of suitable pharmaceuticals are described in "Remington’s Pharmaceutical Sciences". Such compositions will preferably contain an antibody or fragment thereof in a prophylactically or therapeutically effective amount, in a purified form, together with a suitable amount of carrier to provide a form for proper administration to a patient. The formulation should be suitable for the mode of administration, which can be oral, intravenous, intraarterial, buccal, intranasal, aerosolized, bronchial inhalation, or delivery by mechanical ventilation.

[0139] The engineered proteins of the disclosure described herein can be formulated for parenteral administration, e.g., formulated for injection via intradermal, intravenous, intramuscular, subcutaneous, intratumoral, or even intraperitoneal routes. The antibody can alternatively be administered directly to the mucosa by topical routes, e.g., by nasal drops, by inhalation, or by nebulizer. Pharmaceutically acceptable salts include acid salts, and salts formed with inorganic acids such as, for example, hydrochloric acid or phosphoric acid, or organic acids such as acetic acid, oxalic acid, tartaric acid, mandelic acid, and the like. Salts formed with free carboxyl groups can also be derived from inorganic bases such as, for example, sodium, potassium, ammonium, calcium, or ferric hydroxide, and organic bases such as isopropylamine, trimethylamine, 2-ethylaminoethanol, histidine, procaine, and the like.

[0140] Generally, the components of the compositions of the present disclosure are provided either separately or mixed together in unit dosage forms as dry lyophilized powders or anhydrous concentrates in a hermetically sealed container such as an ampoule or sachet, for example, displaying the amount of the active agent. If the composition is to be administered by inhalation, it can be dispensed by an inhalation bottle containing pharmaceutical grade sterile water or saline. If the composition is to be administered by injection, an ampoule of sterile water for injection or saline can be provided so that the components can be mixed prior to administration.

[0141] The compositions of the present disclosure can be formulated in neutral form or in salt form. Pharmaceutically acceptable salts include salts formed with anions such as those derived from hydrochloric acid, phosphoric acid, acetic acid, oxalic acid, tartaric acid, etc., and salts formed with cations such as those derived from sodium, potassium, ammonium, calcium, ferric hydroxide, isopropylamine, triethylamine, 2 - ethylaminoethanol, histidine, procaine, etc.

[0142] Administration can be by a single - dose schedule or a multiple - dose schedule. Multiple - dose schedules can be used in a primary immunization schedule and / or a booster immunization schedule. In a multiple - dose schedule, various doses can be given by the same or different routes. Multiple - dose schedules will typically be administered at intervals of at least one week (e.g., about 2 weeks, about 3 weeks, about 4 weeks, about 6 weeks, about 8 weeks, about 10 weeks, about 12 weeks, about 16 weeks, etc.).

[0143] The compositions disclosed herein can be used to treat both pediatric and adult patients. Thus, the human subject can be less than 1 year old, 1 - 5 years old, 5 - 16 years old, 16 - 55 years old, 55 - 65 years old, or at least 65 years old.

[0144] Preferred routes of administration include, but are not limited to, intramuscular, intraperitoneal, intradermal, subcutaneous, intravenous, intra - arterial, and intraocular injections. Particularly preferred routes of administration include intramuscular, intradermal, and subcutaneous injections.

[0145] V. Immunoassay Methods In yet further embodiments, the disclosure relates to immunoassay methods for binding, purifying, extracting, quantifying, and generally detecting hMPV F protein-binding antibodies in other ways. Such methods can be applied in the conventional sense, but another use is in the quality control and monitoring of vaccine bulk, in which case antibodies can be used to assess the amount or integrity (i.e., long-term stability) of the antigen. Alternatively, this method can be used to screen various antibodies for appropriate / desired reactivity profiles.

[0146] Some immunoassay methods include, to name a few, enzyme-linked immunosorbent assay (ELISA), radioimmunoassay (RIA), immunoradiometric assay, fluorescence immunoassay, chemiluminescence immunoassay, bioluminescence immunoassay, and Western blot. In particular, competitive assays for the detection and quantification of hMPV F protein-binding antibodies are also provided. Generally, an immuno-binding method involves obtaining a sample suspected of containing an hMPV F protein-binding antibody and contacting the sample with an antigen according to the disclosure, optionally under conditions effective to permit the formation of an immune complex.

[0147] These methods include methods for detecting or purifying hMPV F protein-binding antibodies or hMPV F protein from a sample. The antigen is preferably linked to a solid support, such as in the form of a column matrix, and a sample suspected of containing an hMPV F protein-binding antibody is applied to the immobilized antigen. By washing unwanted components from the column, the hMPV F protein-binding antibody that has immunocomplexed to the immobilized antigen remains and is then collected by removing the antigen from the column.

[0148] The immuno-binding method also includes a method for detecting and quantifying the amount of hMPV F protein or related components in a sample, as well as detecting and quantifying any immune complexes formed during the binding process. Here, a sample suspected of containing the hMPV F protein is obtained, the sample is contacted with an antibody that binds to the hMPV F protein or a component thereof, and subsequently the amount of immune complexes formed under specific conditions is detected and quantified. In terms of antigen detection, the biological sample to be analyzed may be any sample suspected of containing the hMPV F protein, including tissue sections or specimens, homogenized tissue extracts, biological fluids (such as nasal washings) including blood and serum, or secretions such as feces or urine.

[0149] Contacting the selected biological sample with the antigen for a sufficient period of time under conditions effective to allow the formation of an immune complex (primary immune complex) generally simply involves adding the antigen composition to the sample and incubating the mixture for a period of time long enough for the antigen to form an immune complex (i.e., bind) with the hMPV F protein-binding antibody. After this time, the sample-antibody composition, such as a tissue section, ELISA plate, dot blot, or Western blot, is generally washed to remove any non-specifically bound antibody species so that only the specifically bound antibody in the primary immune complex is detected.

[0150] Generally, the detection of immune complex formation is well known in the art and can be achieved by the application of a number of approaches. These methods generally rely on the detection of labels or markers such as any of radioactive, fluorescent, biological, and enzyme tags. Patents related to the use of such labels include U.S. Patents 3,817,837, 3,850,752, 3,939,350, 3,996,345, 4,277,437, 4,275,149, and 4,366,241. Of course, as is known in the art, additional advantages can be found through the use of secondary binding ligands such as secondary antibodies and / or biotin / avidin ligand binding arrangements.

[0151] The antibody itself used in the detection may be linked to a detectable label. In that case, the label is then simply detected, whereby the amount of the primary immune complex in the composition is determined. Alternatively, the first antibody that is to be bound within the primary immune complex may be detected using a second binding ligand that has a binding affinity for that antibody. In these cases, the second binding ligand may be linked to a detectable label. The second binding ligand is often itself an antibody and may thus be referred to as a "secondary" antibody. The primary immune complex is contacted with the labeled secondary binding ligand or antibody under conditions effective to allow and for a sufficient period of time to allow the formation of a secondary immune complex. The secondary immune complex is then generally washed to remove any non-specifically bound labeled secondary antibody or ligand, and then the label remaining in the secondary immune complex is detected.

[0152] A further method involves the detection of the primary immune complex by a two-step approach. As described above, a second binding ligand such as an antibody having a binding affinity for the antibody is used to form a secondary immune complex. After washing, the secondary immune complex is contacted with a third binding ligand or antibody having a binding affinity for the second antibody under conditions effective to allow and for a sufficient period of time to allow the formation of a further immune complex (tertiary immune complex). The third ligand or antibody is linked to a detectable label so that the thus formed tertiary immune complex is detected. This system can allow signal amplification if desired.

[0153] One immunoassay method uses two different antibodies. The first biotinylated antibody is used to detect the target antigen, and then the second antibody is used to detect the biotin bound to the complexed biotin. In that method, the sample to be tested is first incubated in a solution containing the antibody of the first step. If the target antigen is present, a portion of the antibody binds to the antigen to form a biotinylated antibody / antigen complex. The antibody / antigen complex is then amplified by incubation in sequential solutions of streptavidin (or avidin), biotinylated DNA, and / or complementary biotinylated DNA, adding additional biotin moieties to the antibody / antigen complex at each step. The amplification step is repeated until a suitable level of amplification is achieved, and when amplification is achieved, the sample is incubated in a solution containing the antibody of the second step against biotin. This second-step antibody is labeled, for example, with an enzyme that can be used to detect the presence of the antibody / antigen complex by histoenzymology using a chromogenic substrate. With suitable amplification, a macroscopically visible conjugate can be produced.

[0154] Another known immunoassay method utilizes the immuno-PCR (polymerase chain reaction) technique. The PCR method is similar to the Cantor method up to incubation with biotinylated DNA, but instead of using multiple streptavidin and biotinylated DNA incubations, the DNA / biotin / streptavidin / antibody complex is washed out with a low pH or high salt buffer that releases the antibody. The resulting wash solution is then used to perform a PCR reaction with suitable primers along with appropriate controls. At least theoretically, the immense amplification capacity and specificity of PCR can be utilized to detect a single antigen molecule.

[0155] 1. ELISA Immunoassays are, in their simplest and most direct sense, binding assays. Certain preferred immunoassays are various types of enzyme-linked immunosorbent assays (ELISAs) and radioimmunoassays (RIAs) known in the art. Immunohistochemical detection using tissue sections is also particularly useful. However, it will be readily understood that the detection is not limited to such techniques, and Western blotting, dot blotting, FACS analysis, etc. may also be used.

[0156] In one exemplary ELISA, an antibody of the present disclosure is immobilized on a selected surface exhibiting protein affinity, such as the wells of a polystyrene microtiter plate. Then, a test composition suspected of containing the hMPV F protein is added to the well. After binding and removing non-specifically bound immune complexes by washing, the bound antigen can be detected. Detection may be achieved by the addition of another anti-hMPV F protein antibody linked to a detectable label. This type of ELISA is a simple "sandwich ELISA". Detection may also be achieved by the addition of a second anti-hMPV F protein antibody, followed by the addition of a third antibody having binding affinity for the second antibody, wherein the third antibody is linked to a detectable label.

[0157] In another exemplary ELISA, a sample suspected of containing the hMPV F protein (e.g., cells that may be infected) is immobilized on the well surface and then contacted with an anti-hMPV F protein antibody of the present disclosure. After binding and removing non-specifically bound immune complexes by washing, the bound anti-hMPV F protein antibody is detected. If the first anti-hMPV F protein antibody is linked to a detectable label, the immune complex may be detected directly. Here too, a second antibody having binding affinity for the first anti-hMPV F protein antibody may be used to detect the immune complex, wherein the second antibody is linked to a detectable label.

[0158] Regardless of the format used, ELISAs share certain specific characteristics, such as coating, incubating, and binding, removing non-specifically bound species by washing, and detecting the bound immune complexes, which are described below.

[0159] When coating the plate with either an antigen or an antibody, generally, the wells of the plate are incubated with a solution of the antigen or antibody for either overnight or a specified period of time. The wells of the plate are then washed to remove incompletely adsorbed material. Next, any remaining available surface of the wells is "coated" with a non-specific protein that is antigenically neutral with respect to the test antiserum. These include solutions of bovine serum albumin (BSA), casein, or non-fat dry milk. Coating enables the blocking of non-specific adsorption sites on the immobilized surface, thus reducing the background caused by non-specific binding of the antiserum to the surface.

[0160] In ELISA, it is probably more common to use secondary or tertiary detection means rather than a direct procedure. Therefore, after binding of the protein or antibody to the well, reducing the background by coating with non-reactive material, and removing unbound material by washing, the immobilized surface is contacted with the biological sample to be tested under conditions effective to allow the formation of immune complexes (antigen / antibody). Detection of the immune complexes then requires a labeled secondary binding ligand or antibody, and a secondary binding ligand or antibody combined with a labeled tertiary antibody or third binding ligand.

[0161] "Conditions effective to allow the formation of immune complexes (antigen / antibody)" preferably means conditions that include diluting the antigen and / or antibody in a solution such as BSA, bovine gamma globulin (BGG), or phosphate buffered saline (PBS) / Tween. These added agents also tend to assist in reducing non-specific background.

[0162] The "suitable" conditions also mean that the incubation is at a temperature sufficient to allow effective binding or over such a period of time. The incubation step is typically about 1 to 2 to 4 hours at a temperature preferably of about 25°C to 27°C, or can be overnight at about 4°C.

[0163] Following all incubation steps in the ELISA, the contact surface is washed to remove uncomplexed material. Preferred washing procedures include washing with a solution such as PBS / Tween or borate buffer. After the formation of specific immune complexes between the test sample and the material initially bound, and subsequent washing, the presence of even trace amounts of immune complexes can be determined.

[0164] To provide a means of detection, the second or third antibody will have an associated label to enable detection. Preferably, this will be an enzyme that produces a color change upon incubation with a suitable chromogenic substrate. Thus, for example, it may be desirable to contact or incubate the first and second immune complexes with urease, glucose oxidase, alkaline phosphatase, or a peroxidase-conjugated antibody for a period of time and under such conditions that are favorable for the development of further immune complex formation (e.g., incubation for 2 hours at room temperature in a PBS-containing solution such as PBS-Tween).

[0165] After incubation with the labeled antibody and removal of unbound material by subsequent washing, the amount of label is quantified, for example, by incubation with a chromogenic substrate such as urea, or bromocresol purple, or 2,2'-azinobis-(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS), or H2O2 (in the case where peroxidase is the enzyme label). Quantification is then achieved, for example, by measuring the degree of color produced using a visible spectrum spectrophotometer.

[0166] 2. Western blot Western blot (alternatively, protein immunoblot) is an analytical technique used to detect specific proteins in a sample of a given tissue homogenate or extract. It uses gel electrophoresis to separate native or denatured proteins either by the length of the polypeptide (denaturing conditions) or by the 3D structure of the protein (native / non-denaturing conditions). The proteins are then transferred to a membrane (typically nitrocellulose or PVDF), where they are probed (detected) using antibodies specific to the target proteins.

[0167] Samples may be taken from whole tissues or from cell cultures. In most cases, solid tissues are first mechanically disrupted using a blender (for larger sample volumes), a homogenizer (for smaller volumes), or sonication. Cells may also be disrupted by one of the mechanical methods described above. Detergents, salts, and buffers in various combinations may be used to facilitate cell lysis and solubilize the proteins. Protease and phosphatase inhibitors are often added to prevent digestion of the sample by enzymes present in the sample itself. Tissue preparation is often carried out at low temperatures to avoid protein denaturation.

[0168] The proteins in the sample are separated using gel electrophoresis. The separation of the proteins may be based on the isoelectric point (pI), molecular weight, charge, or a combination of these factors. The nature of the separation depends on the treatment of the sample and the nature of the gel. This is a very useful method for determining proteins. It is also possible to use two-dimensional (2D) gels that spread the proteins from a single sample in two dimensions. The proteins are separated in the first dimension according to their isoelectric point (the pH at which their net charge is neutral) and in the second dimension according to their molecular weight.

[0169] To make the proteins accessible for antibody detection, they are transferred from the gel onto a membrane made of nitrocellulose or polyvinylidene fluoride (PVDF). The membrane is placed on top of the gel, and a stack of filter papers is placed on top of that. The entire stack is placed in a buffer, and as the buffer carries the proteins with it, they migrate upward through the filter paper by capillary action. Another method for moving the proteins, called electroblotting, uses an electric current to draw the proteins from the gel onto a PVDF or nitrocellulose membrane. The proteins migrate from the gel onto the membrane while maintaining the structure they had within the gel. As a result of this blotting process, the proteins are exposed on the surface monolayer for detection (see below). Both types of membranes are selected for their non-specific protein-binding properties (i.e., they bind equally well to all proteins). Protein binding is based on hydrophobic interactions as well as charge interactions between the membrane and the protein. Nitrocellulose membranes are less expensive than PVDF but are much more fragile and do not withstand repeated probing as well. The uniformity and overall effectiveness of the transfer of proteins from the gel to the membrane can be confirmed by staining the membrane with Coomassie Brilliant Blue or Ponceau S dye. Once transferred, the proteins are detected using a labeled primary antibody or, following an unlabeled primary antibody, indirect detection using a labeled protein A or secondary labeled antibody that binds to the Fc region of the primary antibody.

[0170] 3. Immunohistochemical examination The antibodies of the present disclosure may also be used in conjunction with both fresh frozen tissue blocks and / or formalin-fixed paraffin-embedded tissue blocks prepared for research by immunohistochemical examination (IHC). Methods for preparing tissue blocks from these particulate specimens have been successfully used in previous IHC studies of various prognostic factors and are well known to those skilled in the art (Brown et al., 1990, Abbondanzo et al., 1990, Allred et al., 1990).

[0171] Briefly stated, frozen sections can be prepared by rehydrating 50 ng of frozen "ground" tissue in small plastic capsules at room temperature in phosphate buffered saline (PBS); pelleting the particles by centrifugation; resuspending them in viscous embedding medium (OCT); inverting and / or pelleting the capsules again by centrifugation; snap freezing in -70° C isopentane; cutting away the plastic capsule and / or removing the frozen cylinder from the tissue; fixing the tissue cylinder onto a cryostat microtome chuck; and / or cutting out 25-50 consecutive sections from the capsule. Alternatively, a frozen whole tissue sample may be used for cutting consecutive sections.

[0172] Permanent sections may be prepared by a similar method, which involves rehydrating a 50 mg sample in a plastic microcentrifuge tube; pelleting; resuspending in 10% formalin and fixing for 4 hours; washing / pelleting; resuspending in warm 2.5% agar; pelleting; cooling in ice water to harden the agar; removing the tissue / agar block from the tube; infiltrating the block with paraffin and / or embedding the block in paraffin; and / or cutting out up to 50 permanent consecutive sections. Again, whole tissue samples may be substituted.

[0173] 4. Immunodetection Kit In still further embodiments, the disclosure relates to an immunodetection kit for use with the immunodetection methods described above. Since the hMPV F protein can be detected using an antibody, the antibody may be included in the kit. The immunodetection kit will therefore comprise, in suitable container means, a first antibody that binds to the hMPV F protein and optionally an immunodetection reagent. Alternatively, the hMPV F protein antigen may be used to detect hMPV F protein-binding antibodies. In this case, the immunodetection kit will therefore comprise the hMPV F protein antigen in a suitable container.

[0174] In certain embodiments, the antibody or antigen may be pre - bound to a solid support such as a column matrix and / or the wells of a microtiter plate. The immunodetection reagents of the kit may take any of a variety of forms, including a detectable label associated or linked to the antibody. Also contemplated is a detectable label associated or bound to a secondary binding ligand. An exemplary secondary ligand is a secondary antibody having binding affinity for a first antibody.

[0175] Further suitable immunodetection reagents for use in the kit include a two - component reagent comprising a secondary antibody having binding affinity for the first antibody, together with a third antibody having binding affinity for the second antibody, wherein the third antibody is linked to a detectable label. As described above, several exemplary labels are known in the art and all such labels may be used in connection with the present disclosure.

[0176] The kit may further include a suitably fractionated composition of the hMPV F protein such that it can be used to generate a standard curve for the detection assay, whether the kit is labeled or unlabeled. The kit may contain the antibody - label conjugate in either a complete conjugate form, an intermediate form, or as separate moieties to be conjugated by the user of the kit. The components of the kit may be packaged either in an aqueous medium or in a lyophilized form.

[0177] The container means of the kit will generally include at least one vial, test tube, flask, bottle, syringe, or other container means into which the antibody can be placed, or preferably, suitably fractionated. The kits of the present disclosure will also typically include means for containing the antibody, antigen, and any other reagent containers, tightly sealed for commercial sale. Such containers may include injection - or blow - molded plastic containers in which the desired vials are held.

[0178] 5. Flow Cytometry and FACS The antibodies of the present disclosure may also be used in flow cytometry or FACS. Flow cytometry is a laser or impedance-based technology used in many detection assays, including cell counting, cell sorting, biomarker detection, and protein engineering. This technology allows for the simultaneous multi-parameter analysis of the physical and chemical properties of up to thousands of particles per second by suspending cells in a flowing fluid and passing them through an electrical detection device. Flow cytometry is routinely used in the diagnosis of disorders, but has many other applications in basic research, clinical practice, and clinical trials.

[0179] Fluorescent-activated cell sorting (FACS) is a specialized type of cytometry. It provides a method for sorting a heterogeneous mixture of live cells one cell at a time into two or more containers based on the specific light-scattering and fluorescence properties of each cell. Generally, this technique involves drawing a cell suspension into the center of a narrow, rapidly flowing liquid stream. The flow is arranged so that the cells are widely separated from each other relative to their diameter. A vibrating mechanism divides the cell stream into individual droplets. Just before the flow divides into droplets, the flow passes through a fluorescence measurement station where the fluorescence of each cell is measured. A charged ring is placed just at the point where the flow divides into droplets. Charge is placed on the ring just before the fluorescence intensity is measured, and opposite charge is captured on the droplet as it divides from the flow. The charged droplets then pass through an electrostatic deflection system and fall, with the electrostatic deflection system deflecting the droplets into containers based on their charge.

[0180] In certain embodiments, for use in flow cytometry or FACS, the antibodies of the present disclosure are labeled with a fluorophore and then bound to the cells of interest, which are then analyzed by a flow cytometer or sorted by a FACS machine.

Example

[0181] The following examples are included to illustrate preferred embodiments of the present invention. The techniques disclosed in the examples that follow represent techniques that the inventors have found to function well in the practice of the present invention and, accordingly, can be regarded as constituting a suitable mode for their implementation, which should be understood by those skilled in the art. However, those skilled in the art should understand that, in light of the present disclosure, many modifications can be made to the specific embodiments disclosed without departing from the spirit and scope of the present invention and still obtain the same or similar results.

[0182] Materials and Methods Protein expression and purification. All hMPV F variants were constructed in plasmids containing His and StrepTag II tags by Gibson assembly and confirmed for validity by DNA sequencing. Plasmids encoding the F variant and furin were used in a 4:1 ratio and FreeStyle 293F cells (ThermoFisher) were co-transfected with polyethyleneimine (PEI). Three hours after transfection, kifunensine was added to a final concentration of 5 μM and pluronic F-68 was added to a final concentration of 0.1 v / v% for large-scale transfection. Six days after transfection, the filtered and sterilized supernatant was applied to a StrepTactin column (IBA) for initial purification and then to a Superose 6 10 / 300 or Superdex 200 10 / 300 size exclusion column (SEC) (GE Healthcare) to obtain a monodisperse fraction in SEC buffer (2 mM Tris (pH 8.0), 200 mM NaCl, and 0.03% NaN3). For initial variant screening and characterization, mono-substituted and combinatorial substitution hMPV F variants were purified from 40 mL cell cultures. A Superose 6 16 / 600 column was used to purify the large-scale expression of DS-CavEs2.

[0183] Plasmids encoding the heavy and light chains of MPE8 were co-transfected into FreeStyle 293F cells with PEI at a 1:1 ratio. A stop codon was introduced before the hinge region of the heavy chain to generate the antigen-binding fragment (Fab) of MPE8. To purify MPE8 Fab, the filter-sterilized supernatant was first applied to a CaptureSelect™ IgG-CH1 affinity matrix (ThermoFisher) and then to a Superdex 200 column (GE Healthcare) to obtain a monodisperse fraction in PBS buffer. All protein samples were concentrated to 5 - 10 mg / ml, then snap-frozen in liquid nitrogen and stored at -80 °C.

[0184] Differential scanning calorimetry. Purified hMPV variant at a final concentration of 1 μM was mixed with SYPRO Orange protein gel stain (ThermoFisher) at a final concentration five-fold in a white opaque 96-well plate (VWR). The mixture was then measured using a Roche LightCycler 480 II by continuous fluorescence scanning (λex = 465 nm, λem = 580 nm) with a temperature ramp rate of 4.4 °C / min and a temperature range of 25 °C to 95 °C. Data were plotted as the derivative of the melting curve.

[0185] MPE8 binding analysis by biolayer interferometry. To examine the integrity of the epitopes of hMPV F under various temperature stresses, aliquots of DS-CavEs2 were incubated in a thermocycler at 37 °C, 50 °C, or 70 °C for 30 minutes, or left at 4 °C for 2.5 months, and then tested for MPE8 binding by BLI using an Octet RED96e (ForteBio). Briefly, an anti-human Fab-CH1 second generation (FAB2G) biosensor (ForteBio) was used to capture an equal amount of MPE8 Fab at a concentration of 30 nM in a buffer composed of 10 mM HEPES (pH 7.4), 150 mM NaCl, 0.005 v / v% Tween 20, and 1 mg / ml BSA, and then the biosensor capturing MPE8 was immersed in 50 nM heat-treated DS-CavEs2 to measure the association rate. After a 600-second association step, a 600-second dissociation step was performed in wells containing only buffer. The binding curves were aligned to the baseline and the buffer was subtracted.

[0186] Negative staining EM. The post-fusion hMPV F was heat-treated at 70 °C for 10 minutes and then applied to a CF-400-Cu grid (Electron Microscopy Sciences) that was plasma-cleaned for 45 seconds with O2 / H2 at a 4:1 ratio in a Solarus 950 plasma cleaner (Gatan). The grid was stained using methylamine tungstate (Nanoprobes). The pre-fusion stabilized hMPV F was incubated with a 2-fold molar excess of MPE8 Fab in 1× PBS at room temperature for 30 minutes. The hMPV-F:Fab complex was diluted to a concentration of 0.03 mg / mL in 2 mM Tris (pH 8.0), 200 mM NaCl, and 0.02% NaN3 and then deposited onto a CF-400-Cu grid. The grid was imaged at a magnification of 92,000× (corresponding to a calibrated pixel size of 1.63 Å / pixel) in a Talos F200C TEM microscope (Thermo Fisher Scientific) equipped with a Ceta 16M detector. CTF estimation and particle picking were performed with cisTEM (Grant et al., 2018). The particles were then sent to cryoSPARC v2.15.0 for 2D classification (Punjani et al., 2017).

[0187] X-ray crystallographic analysis of pre-fusion stabilization and complex formation with MPE8. DS-CavEs2 crystals were generated by mixing 500 nl of DS-CavEs2 (10 mg / ml) with 500 nl of a reservoir solution containing 0.1 M MES (pH 6.0) and 12% (v / v) PEG 20k by the hanging-drop vapor diffusion method. The crystals were soaked in a reservoir supplemented with 20% glycerol and frozen in liquid nitrogen. Diffraction data were collected to 2.5 Å at the SBC beamline 19ID (Advanced Photon Source, Argonne National Laboratory). Crystals of DSx2 in complex with MPE8 Fab were grown by mixing 100 nl of the complex (5.4 mg / ml) with 50 nl of a reservoir solution containing 10% (v / v) isopropanol, 0.1 M HEPES (pH 7.5), and 20% (w / v) PEG 4000 by the sitting-drop vapor diffusion method. The crystals were frozen directly in liquid nitrogen without cryoprotectant. Diffraction data for single crystals that diffracted to 2.2 Å were collected at the SBC beamline 19ID (Advanced Photon Source, Argonne National Laboratory). The data were indexed, integrated, and then merged and scaled using Aimless (Evans & Murshudov, 2013) after indexing and integration with iMOSFLM (Battye et al., 2011). Molecular replacement was carried out with Phaser (McCoy et al., 2007), and then the model was subjected to multiple rounds of model building and refined with Coot (Emsley & Cowtan, 2004) and Phenix (Adams et al., 2002), respectively. The statistics for data collection and refinement can be found in Table 2.

[0188] (Table 2) Statistics for crystallographic data collection and refinement TIFF2025102893000005.tif216161The values in parentheses are for the outermost shell with the highest resolution.

[0189] Enzyme-linked immunosorbent assay. A panel of pre-fusion specific monoclonal antibodies against hMPV F (MFP10, Ac967, Ac1025, and MPE8) (Corti et al., 2013) or non-pre-fusion specific antibodies (MF11, MF14) (Battles et al., 2017) were individually immobilized on a 96-well microtiter plate at 4 °C overnight. After a blocking step with 1% BSA in PBS, serial dilutions starting from 4 ng of heat-treated or untreated post-fusion hMPV F were applied to the antibody-coated wells at room temperature for 1 hour. Unbound F was removed by washing three times with 0.1% Tween-20 in PBS. The bound F was then detected by adding an anti-His tag mAb conjugated to horseradish peroxidase (HRP) (Bio-Rad), followed by washing three times with 0.1% Tween-20 in PBS. Then, an HRP substrate (Sigma) was added for color development, and the optical density was read at 492 nm using an ELISA plate reader.

[0190] Example 1 - Structure-based design of pre-fusion stabilized hMPV F Similar to other class I viral fusion glycoproteins, the pre-fusion hMPV F protein exists in a metastable state and readily converts to a stable post-fusion conformation upon induction. To stabilize the F protein in the pre-fusion state, the proline substitution A185P was introduced into the helix-loop-helix region at the trimer apex. This substitution allowed the pre-fusion structure of hMPV to be obtained, but the low expression level of this construct hindered its potential application as a vaccine candidate. Therefore, the H368N substitution (Schowalter et al., 2009), which has previously been shown to increase protein expression levels, similar to the previously described pre-fusion stabilized F protein BV-115 (Battles et al., 2017), was also used. Based on the pre-fusion (PDB ID: 5WB0) and post-fusion (PDB ID: 5L1X) structures of hMPV F, 97 variants were designed from this basic construct, and each variant was then expressed and characterized in terms of production yield, monodispersity, thermal stability, and antigenicity. Exemplary variants are shown in Table 3. The strategies used included disulfide bonds to lock regions that move substantially during the pre- to post-fusion transition, hydrophobic residues to fill internal cavities, polar residues to counter internal charge imbalances, and proline substitutions that are favorable for the pre-fusion conformation and unfavorable for refolding of F1. Regions that move more than 5 Å during the transition are highlighted in blue in Figure 1A (Battles et al., 2017), and the best substitutions from each category are shown in Figure 1B. Overall, 36 variants with single substitutions increased protein expression levels, and many of these variants showed higher thermal stability.

[0191] (Table 3) Exemplary F protein variants TIFF2025102893000006.tif223151TIFF2025102893000007.tif221152TIFF2025102893000008.tif234161TIFF2025102893000009.tif232157TIFF2025102893000010.tif24161

[0192] Example 2 - Single Substitution F Variants The expression profiles of 42 individual variants are summarized in Figure 2A, and the size exclusion chromatography (SEC) traces of selected variants from each design category are shown in Figure 2B. It should be noted that throughout this example, the fold change values of protein yields provided in parentheses are relative to the original values determined when each construct was designed. The values given in Table 3 are from experiments where all constructs were reproduced in parallel and purified, and for each experiment, the area under the curve in the SEC chromatogram was compared to the basic construct control. In the reproduction, all constructs expressed better than in the original expression. The low yield of the basic construct in the original expression was the main factor for the higher yield fold change values in the original values.

[0193] Nine variants with proline substitutions were designed, expressed, and characterized. Six of the nine variants enhanced protein expression (Figure 2A, B). Two variants, A107P and A113P, both located within the fusion peptide, showed 2.9-fold and 1.9-fold increases [5.1-fold and 3.3-fold increases] in protein yield, respectively, compared to the basic construct (Figure 2B). Note that A107P showed a rightward shift of the SEC peak compared to the basic construct, suggesting a more compact trimeric structure (Figure 2B). A344P, the only substitution in domain I (site I) designed to cap the helix, showed a 2-fold increase in protein yield but a slight decrease in Tm by about 0.6 °C. The design of D461P to cap α10 of HRB was also attempted, which resulted in a 1.8-fold increase [2.2-fold increase] in protein yield and a rightward shift of the SEC peak compared to the basic construct, indicating a more compact trimeric structure. Finally, T114P, E146P, and V148P all increased the protein expression level.

[0194] A salt bridge was introduced into the MPV F protein to neutralize internal charge imbalance. L73E and L219K increased the protein expression levels by 1.5-fold and 1.4-fold [2.6-fold and 1.4-fold], respectively (Figs. 2A, B), and both showed longer retention times in SEC. A pair of substitutions L66D / K188R was introduced, which increased the expression level by 1.6-fold. E453Q showed a 1.9-fold [2.8-fold] increase in protein yield, but also showed a significant right shift of the trimer peak in SEC, suggesting that the trimer may be in a relatively closed conformation (Fig. 2C). In addition, several cavity-filling variants showed beneficial effects on the stabilization of the F protein. For example, V231I in domain IIIb (site II) increased the protein expression level by 2.7-fold [3.6-fold] (Figs. 2A, B). Another variant S376T also showed a 2.4-fold increase in protein yield compared to the basic construct. Two other variants S149I and G366S showed 2.3-fold and 1.7-fold [2.3-fold and 2.9-fold] increases in protein expression levels compared to the basic construct and eluted as a monodisperse peak in SEC.

[0195] Regarding the improvement of the protein expression level and thermal stability of hMPV F, pre-fusion stabilizing disulfide bridges were tested. Two examples, L110C / N322C and T365C / V463C, showed an increase in protein yield by 2.5-fold and 1.7-fold [2.8-fold and 2.4-fold] and an increase in Tm by 5.6 °C and 6.2 °C, respectively, compared to the basic construct (Figures 2A, B, C). Variant A116C / A338C also improved the thermal stability compared to the basic construct but did not increase the expression level. On the other hand, A140C / A147C and T127C / N153C, all located in domain IIIa (site V) (i.e., α2, α3, β3, and β4 (Figure 1B)), significantly increased the protein expression level by 3.1-fold and 2.8-fold [6.0-fold and 4.8-fold], respectively (Figures 2A, B). T127C / N153C moderately improved the thermal stability (Figure 2C). The V104C / N457C variant showed a reduction in cleavage by furin compared to the basic construct (Figure 2D), increased Tm by 4 °C, and showed a decrease in the protein expression level (Figures 2B, C). Overall, about 20 out of 42 variants increased the protein expression level (Figure 2A), and 6 of these variants showed an increase in thermal stability.

[0196] Example 3 - Multi-substituted F variants Combinations of single substitutions were introduced into three different variants containing either two disulfide bonds, one disulfide bond and one cavity filling, or one disulfide bond and one salt bridge, generating three all-combination variants. In all three combination variants, the substitution T365C / V463C was included due to its significant improvement in thermal stability. All three variants (T127C / N153C / T365C / V463C, V231I / T365C / V463C, L219K / T365C / V463C) showed further 1.2-fold, 1.9-fold, and 1.2-fold increases in protein yield compared to their parental construct T365C / V463C. The Tm of the variant containing two disulfide bonds increased by 1.7 °C compared to the parental T365C / V463C construct and by 6.4 °C compared to the basic construct (Figure 4A, D). The T127C / N153C / T365C / V463C variant was named DSx2, and either L219K or V231I was further introduced into it. DSx2 / L219K and DSx2 / V231I showed further 1.1-fold and 1.6-fold increases compared to DSx2 (Figure 4B, 6). Furthermore, the variant containing all beneficial mutations (T127C / N153C / T365C / V463C / L219K / V231I) produced 16 mg of protein from 1 L of FreeStyle 293-F cells, showing the highest expression level among all constructs with a further 1.8-fold increase compared to DSx2 and having a Tm of 60.7 °C (Figure 4D, 5). This variant was newly named DS-CavEs, and another disulfide bond design (L110C / N322C) was further introduced into DS-CavEs to generate a penta-substitution variant named MM-1 (T127C / N153C / T365C / V463C / L219K / V231I / L110C / N322C). For MM-1, the protein expression level decreased by 25% compared to DS-CavEs and showed a substantial improvement in Tm (67.6 °C). Another variant named MM-1H (T127C / N153C / T365C / V463C / L219K / V231I / / L110C / N322C / N386H) was generated by reverting H368N to wild-type His368 compared to MM-1.For MM-1H, the protein expression level decreased by 25% compared to DS-CavEs and showed an increased Tm (65.2 °C) (Figures 4C, D). This enhanced thermal stability resulting from the introduction of L110C / N322C may be highly advantageous for vaccine antigens. Due to the benefits shown by the additional disulfide bond, the introduction of A140C / A147C was explored based on its favorable expression profile as a single substitution (Figure 2B). This construct, designated MM-4H, showed a minimal difference in expression yield and a 1.0 °C increase in Tm (Figures 4C, D).

[0197] Furthermore, the A140C / A147C and E453Q substitutions were introduced into MM-1H (which also corresponds to introducing E453Q into MM-4H), and it was designated DS-CavEs2. Large-scale expression of DS-CavEs-2 produced 15.7 mg of prefusion-stabilized F from 1 L of FreeStyle 293-F cells (Figure 4E). The antigenic surface of DS-CavEs2 is well conserved. The affinity of DS-CavEs2 for the prefusion-specific antibody MPE8 (Wen et al., 2017) did not change after incubation at 50 °C for 30 minutes and was equivalent to that of the basic construct (Figure 4F). From nsEM analysis, DS-CavEs2 appears to be a well-folded prefusion trimer, and each protomer was bound by the MPE8 Fab. DS-CavEs2 with additional substitutions enhanced the expression by more than 10-fold and showed the highest thermal stability (Tm 71.8 °C) while maintaining the correct prefusion conformation (Figure 4D), suggesting its potential for vaccine development.

[0198] Example 4 - Crystal Structure of the MPE8-Bound DSx2 Construct For the combinatorial mutant DSx2 construct, to determine the effect of multiple substitutions on the structure of hMPV F, the crystal structure of the protein complexed with the prefusion-specific antibody MPE8 was obtained. This protein complex crystallized in the space group P2 and diffracted X-rays to a resolution of 2.2 Å. After model building and refinement, the structure had R work and R freeIt was first found to have these, and when their structures were further refined, they were improved to 21.3% and 24.2% respectively (Table 2). Compared with the previously determined hMPV F structure (PDBID: 5WB0), DSx2 retained the pre-fusion conformation, and the overall RMSD for 427 Cα residues was 1.8 Å (Figure 9).

[0199] Example 5 - Structure of MPE8-bound DS-CavEs2 The crystal structure of DS-CavEs2 was determined from crystals in the space group P21 to a resolution of 2.5 Å (Table 2). In the absence of MPE8, DS-CavEs2 retained the pre-fusion conformation, with an RMSD of 2.3 Å over 428 Cα atoms shared with PDBID: 5WB0 (Figure 8A). Clear electron density was observed for all disulfide bond substitutions (Cys127 / Cys153, Cys140 / Cys147, Cys110 / Cys322, Cys365 / Cys463) and the cavity filling substitution (I231). Superposition of the membrane-distal half of DS-CavEs2 (sites II, V, and φ) with the previous hMPV F structure (PDBID: 5WB0) revealed a substantial movement of antigenic site IV towards the central three-fold axis (Figure 8A). Superposition of site IV from both structures showed the presence of a rigid body that bends at the center of two long β-strands (β1 and β22) connecting the upper and lower halves of the F protein (Figure 8A). Similar to the DSx2 structure, two disulfide bond substitutions at site V did not change the local conformation. In contrast, the Cys365 / Cys463 substitution pulled the α10 helix away from the central three-fold axis, thus changing the downward trajectory of the HRB (Figure 8A). Negative stain electron microscopy (nsEM) analysis was performed on MPE8 complexed with DS-CavEs2. After 2D class averaging, multiple classes showed DS-CavEs2 as a well-folded pre-fusion trimer bound by two or three MPE8 Fabs, demonstrating that DS-CavEs2 can adopt a trimeric conformation (Figure 8B).

[0200] Example 6 - Pre-fusion stabilized hMPV F variants as immunogens To investigate whether the pre-fusion stabilized hMPV F variants function as immunogens, BALB / c mice are immunized at 3-week intervals with either pre-fusion (e.g., basic construct, DSx2 and DS-CavEs2) or post-fusion F antigens enhanced with CpG. Serum is collected 10 days after the second immunization. The pre-fusion stabilized F constructs are expected to elicit higher neutralizing antibody titers against hMPV A1 and / or hMPV B1 compared to the post-fusion F antigens.

[0201] All of the methods disclosed and claimed herein can be made and executed without undue experimentation in light of the present disclosure. Although the compositions and methods of the invention have been described in terms of preferred embodiments, it will be apparent to those of skill in the art that variations may be applied to the methods and in the steps or in a series of steps of the methods described herein without departing from the concept, spirit, and scope of the invention. More specifically, it will be apparent that certain chemically and physiologically related agents may be substituted for the agents described herein as long as the same or similar results are achieved. All such similar substitutions and modifications apparent to those skilled in the art are deemed to be within the spirit, scope, and concept of the invention as defined by the appended claims.

[0202] References The following references are hereby expressly incorporated by reference herein to the extent that they provide exemplary procedural or other details that supplement the details set forth herein. TIFF2025102893000011.tif202160TIFF2025102893000012.tif238160TIFF2025102893000013.tif238160TIFF2025102893000014.tif48160

[0203] Sequence Information SEQUENCE LISTING <110> Board of Regents, The University of Texas System <120> PREFUSION-STABILIZED HMPV F PROTEINS <150> US 63 / 089,978 <151> 2020-10-09 <160> 17 <170> PatentIn version 3.5 <210> 1 <211> 559 <212> PRT <213> Artificial Sequence <220> <223> BV-115 <400> 1 Met Ser Trp Lys Val Val Ile Ile Phe Ser Leu Leu Ile Thr Pro Gln 1 5 10 15 His Gly Leu Lys Glu Ser Tyr Leu Glu Glu Ser Cys Ser Thr Ile Thr 20 25 30 Glu Gly Tyr Leu Ser Val Leu Arg Thr Gly Trp Tyr Thr Asn Val Phe 35 40 45 Thr Leu Glu Val Gly Asp Val Glu Asn Leu Thr Cys Ala Asp Gly Pro 50 55 60 Ser Leu Ile Lys Thr Glu Leu Asp Leu Thr Lys Ser Ala Leu Arg Glu 65 70 75 80 Leu Arg Thr Val Ser Ala Asp Gln Leu Ala Arg Glu Glu Gln Ile Glu 85 90 95 Asn Pro Arg Arg Arg Arg Phe Val Leu Gly Ala Ile Ala Leu Gly Val 100 105 110 Ala Thr Ala Ala Ala Val Thr Ala Gly Val Ala Ile Ala Lys Thr Ile 115 120 125 Arg Leu Glu Ser Glu Val Thr Ala Ile Lys Asn Ala Leu Lys Lys Thr 130 135 140 Asn Glu Ala Val Ser Thr Leu Gly Asn Gly Val Arg Val Leu Ala Thr 145 150 155 160 Ala Val Arg Glu Leu Lys Asp Phe Val Ser Lys Asn Leu Thr Arg Ala 165 170 175 Ile Asn Lys Asn Lys Cys Asp Ile Pro Asp Leu Lys Met Ala Val Ser 180 185 190 Phe Ser Gln Phe Asn Arg Arg Phe Leu Asn Val Val Arg Gln Phe Ser 195 200 205 Asp Asn Ala Gly Ile Thr Pro Ala Ile Ser Leu Asp Leu Met Thr Asp 210 215 220 Ala Glu Leu Ala Arg Ala Val Ser Asn Met Pro Thr Ser Ala Gly Gln 225 230 235 240 Ile Lys Leu Met Leu Glu Asn Arg Ala Met Val Arg Arg Lys Gly Phe 245 250 255 Gly Ile Leu Ile Gly Val Tyr Gly Ser Ser Val Ile Tyr Met Val Gln 260 265 270 Leu Pro Ile Phe Gly Val Ile Asp Thr Pro Cys Trp Ile Val Lys Ala 275 280 285 Ala Pro Ser Cys Ser Glu Lys Lys Gly Asn Tyr Ala Cys Leu Leu Arg 290 295 300 Glu Asp Gln Gly Trp Tyr Cys Gln Asn Ala Gly Ser Thr Val Tyr Tyr 305 310 315 320 Pro Asn Glu Lys Asp Cys Glu Thr Arg Gly Asp His Val Phe Cys Asp 325 330 335 Thr Ala Ala Gly Ile Asn Val Ala Glu Gln Ser Lys Glu Cys Asn Ile 340 345 350 Asn Ile Ser Thr Thr Asn Tyr Pro Cys Lys Val Ser Thr Gly Arg His 355 360 365 Pro Ile Ser Met Val Ala Leu Ser Pro Leu Gly Ala Leu Val Ala Cys 370 375 380 Tyr Lys Gly Val Ser Cys Ser Ile Gly Ser Asn Arg Val Gly Ile Ile 385 390 395 400 Lys Gln Leu Asn Lys Gly Cys Ser Tyr Ile Thr Asn Gln Asp Ala Asp 405 410 415 Thr Val Thr Ile Asp Asn Thr Val Tyr Gln Leu Ser Lys Val Glu Gly 420 425 430 Glu Gln His Val Ile Lys Gly Arg Pro Val Ser Ser Ser Phe Asp Pro 435 440 445 Val Lys Phe Pro Glu Asp Gln Phe Asn Val Ala Leu Asp Gln Val Phe 450 455 460 Glu Ser Ile Glu Asn Ser Gln Ala Leu Val Asp Gln Ser Asn Arg Ile 465 470 475 480 Leu Ser Ser Ala Glu Lys Gly Asn Thr Ser Gly Arg Glu Asn Leu Tyr 485 490 495 Phe Gln Gly Gly Gly Ser Gly Tyr Ile Pro Glu Ala Pro Arg Asp Gly 500 505 510 Gln Ala Tyr Val Arg Lys Asp Gly Glu Trp Val Leu Leu Ser Thr Phe 515 520 525 Leu Gly Arg Ser Leu Glu Val Leu Phe Gln Gly Pro Gly His His His 530 535 540 His His His His His Ser Ala Trp Ser His Pro Gln Phe Glu Lys 545 550 555 <210> 2 <211> 551 <212> PRT <213> Artificial Sequence <220> <223> JSM-1147 <400> 2 Met Ser Trp Lys Val Val Ile Ile Phe Ser Leu Leu Ile Thr Pro Gln 1 5 10 15 His Gly Leu Lys Glu Ser Tyr Leu Glu Glu Ser Cys Ser Thr Ile Thr 20 25 30 Glu Gly Tyr Leu Ser Val Leu Arg Thr Gly Trp Tyr Thr Asn Val Phe 35 40 45 Thr Leu Glu Val Gly Asp Val Glu Asn Leu Thr Cys Ala Asp Gly Pro 50 55 60 Ser Leu Ile Lys Thr Glu Leu Asp Leu Thr Lys Ser Ala Leu Arg Glu 65 70 75 80 Leu Arg Thr Val Ser Ala Asp Gln Leu Ala Arg Glu Glu Gln Ile Glu 85 90 95 Asn Pro Arg Arg Arg Arg Phe Val Leu Gly Ala Ile Ala Leu Gly Val 100 105 110 Ala Thr Ala Ala Ala Val Thr Ala Gly Val Ala Ile Ala Lys Thr Ile 115 120 125 Arg Leu Glu Ser Glu Val Thr Ala Ile Lys Asn Ala Leu Lys Lys Thr 130 135 140 Asn Glu Ala Val Ser Thr Leu Gly Asn Gly Val Arg Val Leu Ala Thr 145 150 155 160 Ala Val Arg Glu Leu Lys Asp Phe Val Ser Lys Asn Leu Thr Arg Ala 165 170 175 Ile Asn Lys Asn Lys Cys Asp Ile Pro Asp Leu Lys Met Ala Val Ser 180 185 190 Phe Ser Gln Phe Asn Arg Arg Phe Leu Asn Val Val Arg Gln Phe Ser 195 200 205 Asp Asn Ala Gly Ile Thr Pro Ala Ile Ser Leu Asp Leu Met Thr Asp 210 215 220 Ala Glu Leu Ala Arg Ala Val Ser Asn Met Pro Thr Ser Ala Gly Gln 225 230 235 240 Ile Lys Leu Met Leu Glu Asn Arg Ala Met Val Arg Arg Lys Gly Phe 245 250 255 Gly Ile Leu Ile Gly Val Tyr Gly Ser Ser Val Ile Tyr Met Val Gln 260 265 270 Leu Pro Ile Phe Gly Val Ile Asp Thr Pro Cys Trp Ile Val Lys Ala 275 280 285 Ala Pro Ser Cys Ser Glu Lys Lys Gly Asn Tyr Ala Cys Leu Leu Arg 290 295 300 Glu Asp Gln Gly Trp Tyr Cys Gln Asn Ala Gly Ser Thr Val Tyr Tyr 305 310 315 320 Pro Asn Glu Lys Asp Cys Glu Thr Arg Gly Asp His Val Phe Cys Asp 325 330 335 Thr Ala Ala Gly Ile Asn Val Ala Glu Gln Ser Lys Glu Cys Asn Ile 340 345 350 Asn Ile Ser Thr Thr Asn Tyr Pro Cys Lys Val Ser Thr Gly Arg Asn 355 360 365 Pro Ile Ser Met Val Ala Leu Ser Pro Leu Gly Ala Leu Val Ala Cys 370 375 380 Tyr Lys Gly Val Ser Cys Ser Ile Gly Ser Asn Arg Val Gly Ile Ile 385 390 395 400 Lys Gln Leu Asn Lys Gly Cys Ser Tyr Ile Thr Asn Gln Asp Ala Asp 405 410 415 Thr Val Thr Ile Asp Asn Thr Val Tyr Gln Leu Ser Lys Val Glu Gly 420 425 430 Glu Gln His Val Ile Lys Gly Arg Pro Val Ser Ser Ser Phe Asp Pro 435 440 445 Val Lys Phe Pro Glu Asp Gln Phe Asn Val Ala Leu Asp Gln Val Phe 450 455 460 Glu Ser Ile Glu Asn Ser Gln Ala Leu Val Asp Gln Ser Asn Arg Ile 465 470 475 480 Leu Ser Ser Ala Glu Lys Gly Asn Thr Gly Gly Gly Gly Ser Gly Tyr 485 490 495 Ile Pro Glu Ala Pro Arg Asp Gly Gln Ala Tyr Val Arg Lys Asp Gly 500 505 510 Glu Trp Val Leu Leu Ser Thr Phe Leu Gly Arg Ser Leu Glu Val Leu 515 520 525 Phe Gln Gly Pro Gly His His His His His His His His Ser Ala Trp 530 535 540 Ser His Pro Gln Phe Glu Lys 545 550 <210> 3 <211> 542 <212> PRT <213> Artificial Sequence <220> <223> DW-1 <400> 3 Met Ser Trp Lys Val Val Ile Ile Phe Ser Leu Leu Ile Thr Pro Gln 1 5 10 15 His Gly Leu Lys Glu Ser Tyr Leu Glu Glu Ser Cys Ser Thr Ile Thr 20 25 30 Glu Gly Tyr Leu Ser Val Leu Arg Thr Gly Trp Tyr Thr Asn Val Phe 35 40 45 Thr Leu Glu Val Gly Asp Val Glu Asn Leu Thr Cys Ala Asp Gly Pro 50 55 60 Ser Leu Ile Lys Thr Glu Leu Asp Leu Thr Lys Ser Ala Leu Arg Glu 65 70 75 80 Leu Arg Thr Val Ser Ala Asp Gln Leu Ala Arg Glu Glu Gln Ile Glu 85 90 95 Asn Pro Arg Arg Arg Arg Phe Val Leu Gly Ala Ile Ala Leu Gly Val 100 105 110 Ala Thr Ala Ala Ala Val Thr Ala Gly Val Ala Ile Ala Lys Thr Ile 115 120 125 Arg Leu Glu Ser Glu Val Thr Ala Ile Lys Asn Ala Leu Lys Lys Thr 130 135 140 Asn Glu Ala Val Ser Thr Leu Gly Asn Gly Val Arg Val Leu Ala Thr 145 150 155 160 Ala Val Arg Glu Leu Lys Asp Phe Val Ser Lys Asn Leu Thr Arg Ala 165 170 175 Ile Asn Lys Asn Lys Cys Asp Ile Pro Asp Leu Lys Met Ala Val Ser 180 185 190 Phe Ser Gln Phe Asn Arg Arg Phe Leu Asn Val Val Arg Gln Phe Ser 195 200 205 Asp Asn Ala Gly Ile Thr Pro Ala Ile Ser Leu Asp Leu Met Thr Asp 210 215 220 Ala Glu Leu Ala Arg Ala Val Ser Asn Met Pro Thr Ser Ala Gly Gln 225 230 235 240 Ile Lys Leu Met Leu Glu Asn Arg Ala Met Val Arg Arg Lys Gly Phe 245 250 255 Gly Ile Leu Ile Gly Val Tyr Gly Ser Ser Val Ile Tyr Met Val Gln 260 265 270 Leu Pro Ile Phe Gly Val Ile Asp Thr Pro Cys Trp Ile Val Lys Ala 275 280 285 Ala Pro Ser Cys Ser Glu Lys Lys Gly Asn Tyr Ala Cys Leu Leu Arg 290 295 300 Glu Asp Gln Gly Trp Tyr Cys Gln Asn Ala Gly Ser Thr Val Tyr Tyr 305 310 315 320 Pro Asn Glu Lys Asp Cys Glu Thr Arg Gly Asp His Val Phe Cys Asp 325 330 335 Thr Ala Ala Gly Ile Asn Val Ala Glu Gln Ser Lys Glu Cys Asn Ile 340 345 350 Asn Ile Ser Thr Thr Asn Tyr Pro Cys Lys Val Ser Thr Gly Arg His 355 360 365 Pro Ile Ser Met Val Ala Leu Ser Pro Leu Gly Ala Leu Val Ala Cys 370 375 380 Tyr Lys Gly Val Ser Cys Ser Ile Gly Ser Asn Arg Val Gly Ile Ile 385 390 395 400 Lys Gln Leu Asn Lys Gly Cys Ser Tyr Ile Thr Asn Gln Asp Ala Asp 405 410 415 Thr Val Thr Ile Asp Asn Thr Val Tyr Gln Leu Ser Lys Val Glu Gly 420 425 430 Glu Gln His Val Ile Lys Gly Arg Pro Val Ser Ser Ser Phe Asp Pro 435 440 445 Val Lys Phe Pro Glu Asp Gln Phe Asn Val Ala Leu Asp Gln Val Phe 450 455 460 Glu Ser Ile Glu Asn Ser Gln Ala Leu Val Asp Gln Ser Asn Arg Ile 465 470 475 480 Leu Ser Ser Ala Gly Gly Tyr Ile Pro Glu Ala Pro Arg Asp Gly Gln 485 490 495 Ala Tyr Val Arg Lys Asp Gly Glu Trp Val Leu Leu Ser Thr Phe Leu 500 505 510 Gly Arg Ser Leu Glu Val Leu Phe Gln Gly Pro Gly His His His His 515 520 525 His His His His Ser Ala Trp Ser His Pro Gln Phe Glu Lys 530 535 540 <210> 4 <211> 539 <212> PRT <213> Human metapneumovirus <400> 4 Met Ser Trp Lys Val Val Ile Ile Phe Ser Leu Leu Ile Thr Pro Gln 1 5 10 15 His Gly Leu Lys Glu Ser Tyr Leu Glu Glu Ser Cys Ser Thr Ile Thr 20 25 30 Glu Gly Tyr Leu Ser Val Leu Arg Thr Gly Trp Tyr Thr Asn Val Phe 35 40 45 Thr Leu Glu Val Gly Asp Val Glu Asn Leu Thr Cys Ala Asp Gly Pro 50 55 60 Ser Leu Ile Lys Thr Glu Leu Asp Leu Thr Lys Ser Ala Leu Arg Glu 65 70 75 80 Leu Arg Thr Val Ser Ala Asp Gln Leu Ala Arg Glu Glu Gln Ile Glu 85 90 95 Asn Pro Arg Gln Ser Arg Phe Val Leu Gly Ala Ile Ala Leu Gly Val 100 105 110 Ala Thr Ala Ala Ala Val Thr Ala Gly Val Ala Ile Ala Lys Thr Ile 115 120 125 Arg Leu Glu Ser Glu Val Thr Ala Ile Lys Asn Ala Leu Lys Lys Thr 130 135 140 Asn Glu Ala Val Ser Thr Leu Gly Asn Gly Val Arg Val Leu Ala Thr 145 150 155 160 Ala Val Arg Glu Leu Lys Asp Phe Val Ser Lys Asn Leu Thr Arg Ala 165 170 175 Ile Asn Lys Asn Lys Cys Asp Ile Ala Asp Leu Lys Met Ala Val Ser 180 185 190 Phe Ser Gln Phe Asn Arg Arg Phe Leu Asn Val Val Arg Gln Phe Ser 195 200 205 Asp Asn Ala Gly Ile Thr Pro Ala Ile Ser Leu Asp Leu Met Thr Asp 210 215 220 Ala Glu Leu Ala Arg Ala Val Ser Asn Met Pro Thr Ser Ala Gly Gln 225 230 235 240 Ile Lys Leu Met Leu Glu Asn Arg Ala Met Val Arg Arg Lys Gly Phe 245 250 255 Gly Phe Leu Ile Gly Val Tyr Gly Ser Ser Val Ile Tyr Met Val Gln 260 265 270 Leu Pro Ile Phe Gly Val Ile Asp Thr Pro Cys Trp Ile Val Lys Ala 275 280 285 Ala Pro Ser Cys Ser Gly Lys Lys Gly Asn Tyr Ala Cys Leu Leu Arg 290 295 300 Glu Asp Gln Gly Trp Tyr Cys Gln Asn Ala Gly Ser Thr Val Tyr Tyr 305 310 315 320 Pro Asn Glu Lys Asp Cys Glu Thr Arg Gly Asp His Val Phe Cys Asp 325 330 335 Thr Ala Ala Gly Ile Asn Val Ala Glu Gln Ser Lys Glu Cys Asn Ile 340 345 350 Asn Ile Ser Thr Thr Asn Tyr Pro Cys Lys Val Ser Thr Gly Arg His 355 360 365 Pro Ile Ser Met Val Ala Leu Ser Pro Leu Gly Ala Leu Val Ala Cys 370 375 380 Tyr Lys Gly Val Ser Cys Ser Ile Gly Ser Asn Arg Val Gly Ile Ile 385 390 395 400 Lys Gln Leu Asn Lys Gly Cys Ser Tyr Ile Thr Asn Gln Asp Ala Asp 405 410 415 Thr Val Thr Ile Asp Asn Thr Val Tyr Gln Leu Ser Lys Val Glu Gly 420 425 430 Glu Gln His Val Ile Lys Gly Arg Pro Val Ser Ser Ser Phe Asp Pro 435 440 445 Val Lys Phe Pro Glu Asp Gln Phe Asn Val Ala Leu Asp Gln Val Phe 450 455 460 Glu Ser Ile Glu Asn Ser Gln Ala Leu Val Asp Gln Ser Asn Arg Ile 465 470 475 480 Leu Ser Ser Ala Glu Lys Gly Asn Thr Gly Phe Ile Ile Val Ile Ile 485 490 495 Leu Ile Ala Val Leu Gly Ser Thr Met Ile Leu Val Ser Val Phe Ile 500 505 510 Ile Ile Lys Lys Thr Lys Lys Pro Thr Gly Ala Pro Pro Glu Leu Ser 515 520 525 Gly Val Thr Asn Asn Gly Phe Ile Pro His Asn 530 535 <210> 5 <211> 539 <212> PRT <213> Human metapneumovirus <400> 5 Met Ser Trp Lys Val Val Ile Ile Phe Ser Leu Leu Ile Thr Pro Gln 1 5 10 15 His Gly Leu Lys Glu Ser Tyr Leu Glu Glu Ser Cys Ser Thr Ile Thr 20 25 30 Glu Gly Tyr Leu Ser Val Leu Arg Thr Gly Trp Tyr Thr Asn Val Phe 35 40 45 Thr Leu Glu Val Gly Asp Val Glu Asn Leu Thr Cys Ser Asp Gly Pro 50 55 60 Ser Leu Ile Lys Thr Glu Leu Asp Leu Thr Lys Ser Ala Leu Arg Glu 65 70 75 80 Leu Lys Thr Val Ser Ala Asp Gln Leu Ala Arg Glu Glu Gln Ile Glu 85 90 95 Asn Pro Arg Gln Ser Arg Phe Val Leu Gly Ala Ile Ala Leu Gly Val 100 105 110 Ala Thr Ala Ala Ala Val Thr Ala Gly Val Ala Ile Ala Lys Thr Ile 115 120 125 Arg Leu Glu Ser Glu Val Thr Ala Ile Lys Asn Ala Leu Lys Thr Thr 130 135 140 Asn Glu Ala Val Ser Thr Leu Gly Asn Gly Val Arg Val Leu Ala Thr 145 150 155 160 Ala Val Arg Glu Leu Lys Asp Phe Val Ser Lys Asn Leu Thr Arg Ala 165 170 175 Ile Asn Lys Asn Lys Cys Asp Ile Asp Asp Leu Lys Met Ala Val Ser 180 185 190 Phe Ser Gln Phe Asn Arg Arg Phe Leu Asn Val Val Arg Gln Phe Ser 195 200 205 Asp Asn Ala Gly Ile Thr Pro Ala Ile Ser Leu Asp Leu Met Thr Asp 210 215 220 Ala Glu Leu Ala Arg Ala Val Ser Asn Met Pro Thr Ser Ala Gly Gln 225 230 235 240 Ile Lys Leu Met Leu Glu Asn Arg Ala Met Val Arg Arg Lys Gly Phe 245 250 255 Gly Ile Leu Ile Gly Val Tyr Gly Ser Ser Val Ile Tyr Thr Val Gln 260 265 270 Leu Pro Ile Phe Gly Val Ile Asp Thr Pro Cys Trp Ile Val Lys Ala 275 280 285 Ala Pro Ser Cys Ser Glu Lys Lys Gly Asn Tyr Ala Cys Leu Leu Arg 290 295 300 Glu Asp Gln Gly Trp Tyr Cys Gln Asn Ala Gly Ser Thr Val Tyr Tyr 305 310 315 320 Pro Asn Glu Lys Asp Cys Glu Thr Arg Gly Asp His Val Phe Cys Asp 325 330 335 Thr Ala Ala Gly Ile Asn Val Ala Glu Gln Ser Lys Glu Cys Asn Ile 340 345 350 Asn Ile Ser Thr Thr Asn Tyr Pro Cys Lys Val Ser Thr Gly Arg His 355 360 365 Pro Ile Ser Met Val Ala Leu Ser Pro Leu Gly Ala Leu Val Ala Cys 370 375 380 Tyr Lys Gly Val Ser Cys Ser Ile Gly Ser Asn Arg Val Gly Ile Ile 385 390 395 400 Lys Gln Leu Asn Lys Gly Cys Ser Tyr Ile Thr Asn Gln Asp Ala Asp 405 410 415 Thr Val Thr Ile Asp Asn Thr Val Tyr Gln Leu Ser Lys Val Glu Gly 420 425 430 Glu Gln His Val Ile Lys Gly Arg Pro Val Ser Ser Ser Phe Asp Pro 435 440 445 Ile Lys Phe Pro Glu Asp Gln Phe Asn Val Ala Leu Asp Gln Val Phe 450 455 460 Glu Asn Ile Glu Asn Ser Gln Ala Leu Val Asp Gln Ser Asn Arg Ile 465 470 475 480 Leu Ser Ser Ala Glu Lys Gly Asn Thr Gly Phe Ile Ile Val Ile Ile 485 490 495 Leu Ile Ala Val Leu Gly Ser Ser Met Ile Leu Val Ser Ile Phe Ile 500 505 510 Ile Ile Lys Lys Thr Lys Lys Pro Thr Gly Ala Pro Pro Glu Leu Ser 515 520 525 Gly Val Thr Asn Asn Gly Phe Ile Pro His Ser 530 535 <210> 6 <211> 539 <212> PRT <213> Human metapneumovirus <400> 6 Met Ser Trp Lys Val Met Ile Ile Ile Ser Leu Leu Ile Thr Pro Gln 1 5 10 15 His Gly Leu Lys Glu Ser Tyr Leu Glu Glu Ser Cys Ser Thr Ile Thr 20 25 30 Glu Gly Tyr Leu Ser Val Leu Arg Thr Gly Trp Tyr Thr Asn Val Phe 35 40 45 Thr Leu Glu Val Gly Asp Val Glu Asn Leu Thr Cys Thr Asp Gly Pro 50 55 60 Ser Leu Ile Lys Thr Glu Leu Asp Leu Thr Lys Ser Ala Leu Arg Glu 65 70 75 80 Leu Lys Thr Val Ser Ala Asp Gln Leu Ala Arg Glu Glu Gln Ile Glu 85 90 95 Asn Pro Arg Gln Ser Arg Phe Val Leu Gly Ala Ile Ala Leu Gly Val 100 105 110 Ala Thr Ala Ala Ala Val Thr Ala Gly Ile Ala Ile Ala Lys Thr Ile 115 120 125 Arg Leu Glu Ser Glu Val Asn Ala Ile Lys Gly Ala Leu Lys Gln Thr 130 135 140 Asn Glu Ala Val Ser Thr Leu Gly Asn Gly Val Arg Val Leu Ala Thr 145 150 155 160 Ala Val Arg Glu Leu Lys Glu Phe Val Ser Lys Asn Leu Thr Ser Ala 165 170 175 Ile Asn Arg Asn Lys Cys Asp Ile Ala Asp Leu Lys Met Ala Val Ser 180 185 190 Phe Ser Gln Phe Asn Arg Arg Phe Leu Asn Val Val Arg Gln Phe Ser 195 200 205 Asp Asn Ala Gly Ile Thr Pro Ala Ile Ser Leu Asp Leu Met Thr Asp 210 215 220 Ala Glu Leu Ala Arg Ala Val Ser Tyr Met Pro Thr Ser Ala Gly Gln 225 230 235 240 Ile Lys Leu Met Leu Glu Asn Arg Ala Met Val Arg Arg Lys Gly Phe 245 250 255 Gly Ile Leu Ile Gly Val Tyr Gly Ser Ser Val Ile Tyr Met Val Gln 260 265 270 Leu Pro Ile Phe Gly Val Ile Asp Thr Pro Cys Trp Ile Ile Lys Ala 275 280 285 Ala Pro Ser Cys Ser Glu Lys Asn Gly Asn Tyr Ala Cys Leu Leu Arg 290 295 300 Glu Asp Gln Gly Trp Tyr Cys Lys Asn Ala Gly Ser Thr Val Tyr Tyr 305 310 315 320 Pro Asn Glu Lys Asp Cys Glu Thr Arg Gly Asp His Val Phe Cys Asp 325 330 335 Thr Ala Ala Gly Ile Asn Val Ala Glu Gln Ser Arg Glu Cys Asn Ile 340 345 350 Asn Ile Ser Thr Thr Asn Tyr Pro Cys Lys Val Ser Thr Gly Arg His 355 360 365 Pro Ile Ser Met Val Ala Leu Ser Pro Leu Gly Ala Leu Val Ala Cys 370 375 380 Tyr Lys Gly Val Ser Cys Ser Ile Gly Ser Asn Trp Val Gly Ile Ile 385 390 395 400 Lys Gln Leu Pro Lys Gly Cys Ser Tyr Ile Thr Asn Gln Asp Ala Asp 405 410 415 Thr Val Thr Ile Asp Asn Thr Val Tyr Gln Leu Ser Lys Val Glu Gly 420 425 430 Glu Gln His Val Ile Lys Gly Arg Pro Val Ser Ser Ser Phe Asp Pro 435 440 445 Ile Lys Phe Pro Glu Asp Gln Phe Asn Val Ala Leu Asp Gln Val Phe 450 455 460 Glu Ser Ile Glu Asn Ser Gln Ala Leu Val Asp Gln Ser Asn Lys Ile 465 470 475 480 Leu Asn Ser Ala Glu Lys Gly Asn Thr Gly Phe Ile Ile Val Val Ile 485 490 495 Leu Val Ala Val Leu Gly Leu Thr Met Ile Ser Val Ser Ile Ile Ile 500 505 510 Ile Ile Lys Lys Thr Arg Lys Pro Thr Gly Ala Pro Pro Glu Leu Asn 515 520 525 Gly Val Thr Asn Gly Gly Phe Ile Pro His Ser 530 535 <210> 7 <211> 539 <212> PRT <213> Human metapneumovirus <400> 7 Met Ser Trp Lys Val Met Ile Ile Ile Ser Leu Leu Ile Thr Pro Gln 1 5 10 15 His Gly Leu Lys Glu Ser Tyr Leu Glu Glu Ser Cys Ser Thr Ile Thr 20 25 30 Glu Gly Tyr Leu Ser Val Leu Arg Thr Gly Trp Tyr Thr Asn Val Phe 35 40 45 Thr Leu Glu Val Gly Asp Val Glu Asn Leu Thr Cys Thr Asp Gly Pro 50 55 60 Ser Leu Ile Lys Thr Glu Leu Asp Leu Thr Lys Ser Ala Leu Arg Glu 65 70 75 80 Leu Lys Thr Val Ser Ala Asp Gln Leu Ala Arg Glu Glu Gln Ile Glu 85 90 95 Asn Pro Arg Gln Ser Arg Phe Val Leu Gly Ala Ile Ala Leu Gly Val 100 105 110 Ala Thr Ala Ala Ala Val Thr Ala Gly Ile Ala Ile Ala Lys Thr Ile 115 120 125 Arg Leu Glu Ser Glu Val Asn Ala Ile Lys Gly Ala Leu Lys Thr Thr 130 135 140 Asn Glu Ala Val Ser Thr Leu Gly Asn Gly Val Arg Val Leu Ala Thr 145 150 155 160 Ala Val Arg Glu Leu Lys Glu Phe Val Ser Lys Asn Leu Thr Ser Ala 165 170 175 Ile Asn Lys Asn Lys Cys Asp Ile Ala Asp Leu Lys Met Ala Val Ser 180 185 190 Phe Ser Gln Phe Asn Arg Arg Phe Leu Asn Val Val Arg Gln Phe Ser 195 200 205 Asp Asn Ala Gly Ile Thr Pro Ala Ile Ser Leu Asp Leu Met Thr Asp 210 215 220 Ala Glu Leu Ala Arg Ala Val Ser Tyr Met Pro Thr Ser Ala Gly Gln 225 230 235 240 Ile Lys Leu Met Leu Glu Asn Arg Ala Met Val Arg Arg Lys Gly Phe 245 250 255 Gly Ile Leu Ile Gly Val Tyr Gly Ser Ser Val Ile Tyr Met Val Gln 260 265 270 Leu Pro Ile Phe Gly Val Ile Asp Thr Pro Cys Trp Ile Ile Lys Ala 275 280 285 Ala Pro Ser Cys Ser Glu Lys Asp Gly Asn Tyr Ala Cys Leu Leu Arg 290 295 300 Glu Asp Gln Gly Trp Tyr Cys Lys Asn Ala Gly Ser Thr Val Tyr Tyr 305 310 315 320 Pro Asn Asp Lys Asp Cys Glu Thr Arg Gly Asp His Val Phe Cys Asp 325 330 335 Thr Ala Ala Gly Ile Asn Val Ala Glu Gln Ser Arg Glu Cys Asn Ile 340 345 350 Asn Ile Ser Thr Thr Asn Tyr Pro Cys Lys Val Ser Thr Gly Arg His 355 360 365 Pro Ile Ser Met Val Ala Leu Ser Pro Leu Gly Ala Leu Val Ala Cys 370 375 380 Tyr Lys Gly Val Ser Cys Ser Ile Gly Ser Asn Arg Val Gly Ile Ile 385 390 395 400 Lys Gln Leu Pro Lys Gly Cys Ser Tyr Ile Thr Asn Gln Asp Ala Asp 405 410 415 Thr Val Thr Ile Asp Asn Thr Val Tyr Gln Leu Ser Lys Val Glu Gly 420 425 430 Glu Gln His Val Ile Lys Gly Arg Pro Val Ser Ser Ser Phe Asp Pro 435 440 445 Ile Lys Phe Pro Glu Asp Gln Phe Asn Val Ala Leu Asp Gln Val Phe 450 455 460 Glu Ser Ile Glu Asn Ser Gln Ala Leu Val Asp Gln Ser Asn Lys Ile 465 470 475 480 Leu Asn Ser Ala Glu Lys Gly Asn Thr Gly Phe Ile Ile Val Ile Ile 485 490 495 Leu Ile Ala Val Leu Gly Leu Thr Met Ile Ser Val Ser Ile Ile Ile 500 505 510 Ile Ile Lys Lys Thr Arg Lys Pro Thr Gly Ala Pro Pro Glu Leu Asn 515 520 525 Gly Val Thr Asn Gly Gly Phe Ile Pro His Ser 530 535 <210> 8 <211> 11 <212> PRT <213> Artificial Sequence <220> <223> Synthetic polypeptide <400> 8 Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Arg 1 5 10 <210> 9 <211> 4 <212> PRT <213> Artificial Sequence <220> <223> Artificial polypeptide sequence <400> 9 Arg Gln Ser Arg 1 <210> 10 <211> 4 <212> PRT <213> Artificial Sequence <220> <223> Artificial polypeptide sequence <400> 10 Arg Arg Arg Arg 1 <210> 11 <211> 18 <212> PRT <213> Artificial Sequence <220> <223> Artificial polypeptide sequence <400> 11 Met Ser Trp Lys Val Met Ile Ile Ile Ser Leu Leu Ile Thr Pro Gln 1 5 10 15 His Gly <210> 12 <211> 6 <212> PRT <213> Artificial Sequence <220> <223> Artificial polypeptide sequence <400> 12 Gly Gly Ser Gly Gly Ser 1 5 <210> 13 <211> 6 <212> PRT <213> Artificial Sequence <220> <223> Artificial polypeptide sequence <400> 13 Gly Gly Gly Gly Gly Gly 1 5 <210> 14 <211> 454 <212> PRT <213> Artificial Sequence <220> <223> DS-CavEs <400> 14 Leu Lys Glu Ser Tyr Leu Glu Glu Ser Cys Ser Thr Ile Thr Glu Gly 1 5 10 15 Tyr Leu Ser Val Leu Arg Thr Gly Trp Tyr Thr Asn Val Phe Thr Leu 20 25 30 Glu Val Gly Asp Val Glu Asn Leu Thr Cys Ala Asp Gly Pro Ser Leu 35 40 45 Ile Lys Thr Glu Leu Asp Leu Thr Lys Ser Ala Leu Arg Glu Leu Arg 50 55 60 Thr Val Ser Ala Asp Gln Leu Ala Arg Glu Glu Gln Ile Glu Asn Pro 65 70 75 80 Arg Arg Arg Arg Phe Val Leu Gly Ala Ile Ala Leu Gly Val Ala Thr 85 90 95 Ala Ala Ala Val Thr Ala Gly Val Ala Ile Ala Lys Cys Ile Arg Leu 100 105 110 Glu Ser Glu Val Thr Ala Ile Lys Asn Ala Leu Lys Lys Thr Asn Glu 115 120 125 Ala Val Ser Thr Leu Gly Cys Gly Val Arg Val Leu Ala Thr Ala Val 130 135 140 Arg Glu Leu Lys Asp Phe Val Ser Lys Asn Leu Thr Arg Ala Ile Asn 145 150 155 160 Lys Asn Lys Cys Asp Ile Pro Asp Leu Lys Met Ala Val Ser Phe Ser 165 170 175 Gln Phe Asn Arg Arg Phe Leu Asn Val Val Arg Gln Phe Ser Asp Asn 180 185 190 Ala Gly Ile Thr Pro Ala Ile Ser Lys Asp Leu Met Thr Asp Ala Glu 195 200 205 Leu Ala Arg Ala Ile Ser Asn Met Pro Thr Ser Ala Gly Gln Ile Lys 210 215 220 Leu Met Leu Glu Asn Arg Ala Met Val Arg Arg Lys Gly Phe Gly Ile 225 230 235 240 Leu Ile Gly Val Tyr Gly Ser Ser Val Ile Tyr Met Val Gln Leu Pro 245 250 255 Ile Phe Gly Val Ile Asp Thr Pro Cys Trp Ile Val Lys Ala Ala Pro 260 265 270 Ser Cys Ser Glu Lys Lys Gly Asn Tyr Ala Cys Leu Leu Arg Glu Asp 275 280 285 Gln Gly Trp Tyr Cys Gln Asn Ala Gly Ser Thr Val Tyr Tyr Pro Asn 290 295 300 Glu Lys Asp Cys Glu Thr Arg Gly Asp His Val Phe Cys Asp Thr Ala 305 310 315 320 Ala Gly Ile Asn Val Ala Glu Gln Ser Lys Glu Cys Asn Ile Asn Ile 325 330 335 Ser Thr Thr Asn Tyr Pro Cys Lys Val Ser Cys Gly Arg Asn Pro Ile 340 345 350 Ser Met Val Ala Leu Ser Pro Leu Gly Ala Leu Val Ala Cys Tyr Lys 355 360 365 Gly Val Ser Cys Ser Ile Gly Ser Asn Arg Val Gly Ile Ile Lys Gln 370 375 380 Leu Asn Lys Gly Cys Ser Tyr Ile Thr Asn Gln Asp Ala Asp Thr Val 385 390 395 400 Thr Ile Asp Asn Thr Val Tyr Gln Leu Ser Lys Val Glu Gly Glu Gln 405 410 415 His Val Ile Lys Gly Arg Pro Val Ser Ser Ser Phe Asp Pro Val Lys 420 425 430 Phe Pro Glu Asp Gln Phe Asn Val Ala Leu Asp Gln Cys Phe Glu Ser 435 440 445 Ile Glu Asn Ser Gln Ala 450 <210> 15 <211> 454 <212> PRT <213> Artificial Sequence <220> <223> DS-CavEs2 <400> 15 Leu Lys Glu Ser Tyr Leu Glu Glu Ser Cys Ser Thr Ile Thr Glu Gly 1 5 10 15 Tyr Leu Ser Val Leu Arg Thr Gly Trp Tyr Thr Asn Val Phe Thr Leu 20 25 30 Glu Val Gly Asp Val Glu Asn Leu Thr Cys Ala Asp Gly Pro Ser Leu 35 40 45 Ile Lys Thr Glu Leu Asp Leu Thr Lys Ser Ala Leu Arg Glu Leu Arg 50 55 60 Thr Val Ser Ala Asp Gln Leu Ala Arg Glu Glu Gln Ile Glu Asn Pro 65 70 75 80 Arg Arg Arg Arg Phe Val Leu Gly Ala Ile Ala Cys Gly Val Ala Thr 85 90 95 Ala Ala Ala Val Thr Ala Gly Val Ala Ile Ala Lys Cys Ile Arg Leu 100 105 110 Glu Ser Glu Val Thr Ala Ile Lys Asn Cys Leu Lys Lys Thr Asn Glu 115 120 125 Cys Val Ser Thr Leu Gly Cys Gly Val Arg Val Leu Ala Thr Ala Val 130 135 140 Arg Glu Leu Lys Asp Phe Val Ser Lys Asn Leu Thr Arg Ala Ile Asn 145 150 155 160 Lys Asn Lys Cys Asp Ile Pro Asp Leu Lys Met Ala Val Ser Phe Ser 165 170 175 Gln Phe Asn Arg Arg Phe Leu Asn Val Val Arg Gln Phe Ser Asp Asn 180 185 190 Ala Gly Ile Thr Pro Ala Ile Ser Lys Asp Leu Met Thr Asp Ala Glu 195 200 205 Leu Ala Arg Ala Ile Ser Asn Met Pro Thr Ser Ala Gly Gln Ile Lys 210 215 220 Leu Met Leu Glu Asn Arg Ala Met Val Arg Arg Lys Gly Phe Gly Ile 225 230 235 240 Leu Ile Gly Val Tyr Gly Ser Ser Val Ile Tyr Met Val Gln Leu Pro 245 250 255 Ile Phe Gly Val Ile Asp Thr Pro Cys Trp Ile Val Lys Ala Ala Pro 260 265 270 Ser Cys Ser Glu Lys Lys Gly Asn Tyr Ala Cys Leu Leu Arg Glu Asp 275 280 285 Gln Gly Trp Tyr Cys Gln Asn Ala Gly Ser Thr Val Tyr Tyr Pro Cys 290 295 300 Glu Lys Asp Cys Glu Thr Arg Gly Asp His Val Phe Cys Asp Thr Ala 305 310 315 320 Ala Gly Ile Asn Val Ala Glu Gln Ser Lys Glu Cys Asn Ile Asn Ile 325 330 335 Ser Thr Thr Asn Tyr Pro Cys Lys Val Ser Cys Gly Arg His Pro Ile 340 345 350 Ser Met Val Ala Leu Ser Pro Leu Gly Ala Leu Val Ala Cys Tyr Lys 355 360 365 Gly Val Ser Cys Ser Ile Gly Ser Asn Arg Val Gly Ile Ile Lys Gln 370 375 380 Leu Asn Lys Gly Cys Ser Tyr Ile Thr Asn Gln Asp Ala Asp Thr Val 385 390 395 400 Thr Ile Asp Asn Thr Val Tyr Gln Leu Ser Lys Val Glu Gly Glu Gln 405 410 415 His Val Ile Lys Gly Arg Pro Val Ser Ser Ser Phe Asp Pro Val Lys 420 425 430 Phe Pro Gln Asp Gln Phe Asn Val Ala Leu Asp Gln Cys Phe Glu Ser 435 440 445 Ile Glu Asn Ser Gln Ala 450 <210> 16 <211> 454 <212> PRT <213> Artificial Sequence <220> <223> Artificial polypeptide sequence <220> <221> MISC_FEATURE <222> (79)..(84) <223> Xaa is Gly or Ser <400> 16 Leu Lys Glu Ser Tyr Leu Glu Glu Ser Cys Ser Thr Ile Thr Glu Gly 1 5 10 15 Tyr Leu Ser Val Leu Arg Thr Gly Trp Tyr Thr Asn Val Phe Thr Leu 20 25 30 Glu Val Gly Asp Val Glu Asn Leu Thr Cys Ala Asp Gly Pro Ser Leu 35 40 45 Ile Lys Thr Glu Leu Asp Leu Thr Lys Ser Ala Leu Arg Glu Leu Arg 50 55 60 Thr Val Ser Ala Asp Gln Leu Ala Arg Glu Glu Gln Ile Glu Xaa Xaa 65 70 75 80 Xaa Xaa Xaa Xaa Phe Val Leu Gly Ala Ile Ala Leu Gly Val Ala Thr 85 90 95 Ala Ala Ala Val Thr Ala Gly Val Ala Ile Ala Lys Cys Ile Arg Leu 100 105 110 Glu Ser Glu Val Thr Ala Ile Lys Asn Ala Leu Lys Lys Thr Asn Glu 115 120 125 Ala Val Ser Thr Leu Gly Cys Gly Val Arg Val Leu Ala Thr Ala Val 130 135 140 Arg Glu Leu Lys Asp Phe Val Ser Lys Asn Leu Thr Arg Ala Ile Asn 145 150 155 160 Lys Asn Lys Cys Asp Ile Pro Asp Leu Lys Met Ala Val Ser Phe Ser 165 170 175 Gln Phe Asn Arg Arg Phe Leu Asn Val Val Arg Gln Phe Ser Asp Asn 180 185 190 Ala Gly Ile Thr Pro Ala Ile Ser Lys Asp Leu Met Thr Asp Ala Glu 195 200 205 Leu Ala Arg Ala Ile Ser Asn Met Pro Thr Ser Ala Gly Gln Ile Lys 210 215 220 Leu Met Leu Glu Asn Arg Ala Met Val Arg Arg Lys Gly Phe Gly Ile 225 230 235 240 Leu Ile Gly Val Tyr Gly Ser Ser Val Ile Tyr Met Val Gln Leu Pro 245 250 255 Ile Phe Gly Val Ile Asp Thr Pro Cys Trp Ile Val Lys Ala Ala Pro 260 265 270 Ser Cys Ser Glu Lys Lys Gly Asn Tyr Ala Cys Leu Leu Arg Glu Asp 275 280 285 Gln Gly Trp Tyr Cys Gln Asn Ala Gly Ser Thr Val Tyr Tyr Pro Asn 290 295 300 Glu Lys Asp Cys Glu Thr Arg Gly Asp His Val Phe Cys Asp Thr Ala 305 310 315 320 Ala Gly Ile Asn Val Ala Glu Gln Ser Lys Glu Cys Asn Ile Asn Ile 325 330 335 Ser Thr Thr Asn Tyr Pro Cys Lys Val Ser Cys Gly Arg Asn Pro Ile 340 345 350 Ser Met Val Ala Leu Ser Pro Leu Gly Ala Leu Val Ala Cys Tyr Lys 355 360 365 Gly Val Ser Cys Ser Ile Gly Ser Asn Arg Val Gly Ile Ile Lys Gln 370 375 380 Leu Asn Lys Gly Cys Ser Tyr Ile Thr Asn Gln Asp Ala Asp Thr Val 385 390 395 400 Thr Ile Asp Asn Thr Val Tyr Gln Leu Ser Lys Val Glu Gly Glu Gln 405 410 415 His Val Ile Lys Gly Arg Pro Val Ser Ser Ser Phe Asp Pro Val Lys 420 425 430 Phe Pro Glu Asp Gln Phe Asn Val Ala Leu Asp Gln Cys Phe Glu Ser 435 440 445 Ile Glu Asn Ser Gln Ala 450 <210> 17 <211> 454 <212> PRT <213> Artificial Sequence <220> <223> Artificial polypeptide sequence <220> <221> MISC_FEATURE <222> (79)..(84) <223> Xaa is Gly or Ser <400> 17 Leu Lys Glu Ser Tyr Leu Glu Glu Ser Cys Ser Thr Ile Thr Glu Gly 1 5 10 15 Tyr Leu Ser Val Leu Arg Thr Gly Trp Tyr Thr Asn Val Phe Thr Leu 20 25 30 Glu Val Gly Asp Val Glu Asn Leu Thr Cys Ala Asp Gly Pro Ser Leu 35 40 45 Ile Lys Thr Glu Leu Asp Leu Thr Lys Ser Ala Leu Arg Glu Leu Arg 50 55 60 Thr Val Ser Ala Asp Gln Leu Ala Arg Glu Glu Gln Ile Glu Xaa Xaa 65 70 75 80 Xaa Xaa Xaa Xaa Phe Val Leu Gly Ala Ile Ala Cys Gly Val Ala Thr 85 90 95 Ala Ala Ala Val Thr Ala Gly Val Ala Ile Ala Lys Cys Ile Arg Leu 100 105 110 Glu Ser Glu Val Thr Ala Ile Lys Asn Cys Leu Lys Lys Thr Asn Glu 115 120 125 Cys Val Ser Thr Leu Gly Cys Gly Val Arg Val Leu Ala Thr Ala Val 130 135 140 Arg Glu Leu Lys Asp Phe Val Ser Lys Asn Leu Thr Arg Ala Ile Asn 145 150 155 160 Lys Asn Lys Cys Asp Ile Pro Asp Leu Lys Met Ala Val Ser Phe Ser 165 170 175 Gln Phe Asn Arg Arg Phe Leu Asn Val Val Arg Gln Phe Ser Asp Asn 180 185 190 Ala Gly Ile Thr Pro Ala Ile Ser Lys Asp Leu Met Thr Asp Ala Glu 195 200 205 Leu Ala Arg Ala Ile Ser Asn Met Pro Thr Ser Ala Gly Gln Ile Lys 210 215 220 Leu Met Leu Glu Asn Arg Ala Met Val Arg Arg Lys Gly Phe Gly Ile 225 230 235 240 Leu Ile Gly Val Tyr Gly Ser Ser Val Ile Tyr Met Val Gln Leu Pro 245 250 255 Ile Phe Gly Val Ile Asp Thr Pro Cys Trp Ile Val Lys Ala Ala Pro 260 265 270 Ser Cys Ser Glu Lys Lys Gly Asn Tyr Ala Cys Leu Leu Arg Glu Asp 275 280 285 Gln Gly Trp Tyr Cys Gln Asn Ala Gly Ser Thr Val Tyr Tyr Pro Cys 290 295 300 Glu Lys Asp Cys Glu Thr Arg Gly Asp His Val Phe Cys Asp Thr Ala 305 310 315 320 Ala Gly Ile Asn Val Ala Glu Gln Ser Lys Glu Cys Asn Ile Asn Ile 325 330 335 Ser Thr Thr Asn Tyr Pro Cys Lys Val Ser Cys Gly Arg His Pro Ile 340 345 350 Ser Met Val Ala Leu Ser Pro Leu Gly Ala Leu Val Ala Cys Tyr Lys 355 360 365 Gly Val Ser Cys Ser Ile Gly Ser Asn Arg Val Gly Ile Ile Lys Gln 370 375 380 Leu Asn Lys Gly Cys Ser Tyr Ile Thr Asn Gln Asp Ala Asp Thr Val 385 390 395 400 Thr Ile Asp Asn Thr Val Tyr Gln Leu Ser Lys Val Glu Gly Glu Gln 405 410 415 His Val Ile Lys Gly Arg Pro Val Ser Ser Ser Phe Asp Pro Val Lys 420 425 430 Phe Pro Gln Asp Gln Phe Asn Val Ala Leu Asp Gln Cys Phe Glu Ser 435 440 445 Ile Glu Asn Ser Gln Ala 450

Claims

**Claim 1** A nucleic acid molecule comprising a nucleotide sequence encoding an amino acid sequence of an engineered protein comprising an external domain of a metapneumovirus (MPV) F protein having at least 90% identity to any one of amino acids 19 to 489 of SEQ ID NOs: 4-7, wherein the engineered protein comprises engineered disulfide bonds comprising pairs of cysteine substitutions corresponding to T127C and N153C, and / or L110C and N322C, with respect to any one of the sequences of SEQ ID NOs: 4-7, and wherein the nucleic acid molecule is an mRNA molecule. **Claim 2** The nucleic acid molecule of claim 1, wherein the engineered protein further comprises a substitution at a position corresponding to L219 and / or V231. **Claim 3** The nucleic acid molecule of claim 1, wherein the engineered protein further comprises substitutions corresponding to L219K and V231I. **Claim 4** The nucleic acid molecule of claim 1, wherein the engineered protein further comprises a pair of cysteine substitutions corresponding to T365C and V463C. **Claim 5** The nucleic acid molecule of claim 1, wherein the engineered protein comprises a pair of cysteine substitutions corresponding to T127C and N153C, a pair of cysteine substitutions corresponding to T365C and V463C, and a substitution corresponding to L219K. **Claim 6** The nucleic acid molecule of claim 1, wherein the engineered protein comprises a pair of cysteine substitutions corresponding to T127C and N153C, a pair of cysteine substitutions corresponding to T365C and V463C, and a substitution corresponding to V231I. **Claim 7** The nucleic acid molecule of claim 1, wherein the engineered protein further comprises a proline substitution corresponding to A / D185P. **Claim 8** The nucleic acid molecule of claim 1, wherein the engineered protein further comprises a substitution from a residue corresponding to any one of positions 87 to 104 of SEQ ID NOs: 4-7 to GGGSGGSGGSR (SEQ ID NO: 8). **Claim 9** The nucleic acid molecule of claim 1, wherein the engineered protein further comprises electrostatic interaction substitutions at positions corresponding to L66, L73, N145, Q195, E453, K188, H368, D461, T49, and / or V262. **Claim 10** The nucleic acid molecule according to claim 9, wherein the electrostatic interaction substitution comprises a substitution corresponding to L66N, L73E, N145E, Q195K, E453Q, L66D, K188R, H368R, D461E, T49E, and / or V262D.

11. The nucleic acid molecule according to claim 1, wherein the engineered protein comprises substitutions corresponding to T127C, N153C, T365C, V463C, L219K, and V231I.

12. The nucleic acid molecule according to claim 1, wherein the engineered protein comprises substitutions corresponding to T127C, N153C, A185P, T365C, V463C, L219K, and V231I.

13. The nucleic acid molecule according to claim 1, wherein the engineered protein comprises substitutions corresponding to T127C, N153C, A185P, T365C, V463C, L219K, V231I, and a substitution from RQSR (residues corresponding to positions 99 - 102 of any one of SEQ ID NOs: 4 - 7) to RRRR (SEQ ID NO: 10).

14. The nucleic acid molecule according to claim 1, wherein the engineered protein comprises substitutions corresponding to L110C, T127C, A140C, A147C, N153C, L219K, V231I, N322C, T365C, E453Q, and V463C.

15. The nucleic acid molecule according to claim 1, wherein the engineered protein comprises substitutions corresponding to L110C, T127C, A140C, A147C, N153C, A185P, L219K, V231I, N322C, T365C, E453Q, and V463C.

16. The nucleic acid molecule according to claim 1, wherein the engineered protein comprises substitutions corresponding to L110C, T127C, A140C, A147C, N153C, A185P, L219K, V231I, N322C, T365C, E453Q, V463C, and a substitution from RQSR (residues corresponding to positions 99 - 102 of any one of SEQ ID NOs: 4 - 7) to RRRR (SEQ ID NO: 10).

17. The nucleic acid molecule according to claim 1, wherein the engineered protein comprises a polypeptide sequence that is at least 95% identical to SEQ ID NO:

14.

18. The nucleic acid molecule according to claim 1, wherein the engineered protein comprises a polypeptide sequence that is at least 95% identical to SEQ ID NO:

15.

19. The nucleic acid molecule according to claim 1, wherein the engineered protein comprises a polypeptide sequence having at least 95% identity to the amino acid sequence of SEQ ID NO:

2.

20. The nucleic acid molecule according to claim 1, wherein the engineered protein comprises an hMPV F protein extracellular domain having at least 95% identity to amino acids 19-489 of SEQ ID NO:

4.

21. The nucleic acid molecule according to claim 1, wherein the engineered protein is fused to a trimerization domain.

22. The nucleic acid molecule according to claim 21, wherein the trimerization domain comprises the trimerization domain of T4 fibritin.

23. The nucleic acid molecule according to claim 1, wherein the engineered protein is fused to a transmembrane domain.

24. The nucleic acid molecule according to claim 1, wherein the engineered protein comprises an N-terminal signal sequence.

25. A pharmaceutical composition comprising a pharmaceutically acceptable carrier and the nucleic acid molecule according to claim 1.

26. The pharmaceutical composition according to claim 25, further comprising an adjuvant.

27. A pharmaceutical composition for preventing metapneumovirus (MPV) infection or a disease associated with MPV infection in a subject, the pharmaceutical composition comprising the nucleic acid molecule according to claim 1.

28. A nucleic acid molecule, which is an mRNA molecule, comprising a nucleotide sequence encoding the amino acid sequence of an engineered protein comprising the extracellular domain of the metapneumovirus (MPV) F protein having the amino acid sequence of SEQ ID NO: 15.

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